BCFTOOLS(1) BCFTOOLS(1) NAME bcftools - utilities for variant calling and manipulating VCFs and BCFs. SYNOPSIS bcftools [--version|--version-only] [--help] [COMMAND] [OPTIONS] DESCRIPTION BCFtools is a set of utilities that manipulate variant calls in the Vari- ant Call Format (VCF) and its binary counterpart BCF. All commands work transparently with both VCFs and BCFs, both uncompressed and BGZF-com- pressed. Most commands accept VCF, bgzipped VCF and BCF with filetype detected auto- matically even when streaming from a pipe. Indexed VCF and BCF will work in all situations. Un-indexed VCF and BCF and streams will work in most, but not all situations. In general, whenever multiple VCFs are read simultane- ously, they must be indexed and therefore also compressed. (Note that files with non-standard index names can be accessed as e.g. "bcftools view -r X:2928329 file.vcf.gz##idx##non-standard-index-name".) BCFtools is designed to work on a stream. It regards an input file "-" as the standard input (stdin) and outputs to the standard output (stdout). Several commands can thus be combined with Unix pipes. VERSION This manual page was last updated 2026-03-18 and refers to bcftools git version 1.23.1. BCF1 The obsolete BCF1 format output by versions of samtools <= 0.1.19 is not compatible with this version of bcftools. To read BCF1 files one can use the view command from old versions of bcftools packaged with samtools ver- sions <= 0.1.19 to convert to VCF, which can then be read by this version of bcftools. samtools-0.1.19/bcftools/bcftools view file.bcf1 | bcftools view VARIANT CALLING See bcftools call for variant calling from the output of the samtools mpileup command. In versions of samtools <= 0.1.19 calling was done with bcftools view. Users are now required to choose between the old samtools calling model (-c/--consensus-caller) and the new multiallelic calling model (-m/--multiallelic-caller). The multiallelic calling model is recom- mended for most tasks. FILTERING EXPRESSIONS See EXPRESSIONS LIST OF COMMANDS For a full list of available commands, run bcftools without arguments. For a full list of available options, run bcftools COMMAND without arguments. * annotate .. edit VCF files, add or remove annotations * call .. SNP/indel calling (former "view") * cnv .. Copy Number Variation caller * concat .. concatenate VCF/BCF files from the same set of samples * consensus .. create consensus sequence by applying VCF variants * convert .. convert VCF/BCF to other formats and back * csq .. haplotype aware consequence caller * filter .. filter VCF/BCF files using fixed thresholds * gtcheck .. check sample concordance, detect sample swaps and contami- nation * head .. view VCF/BCF file headers * index .. index VCF/BCF * isec .. intersections of VCF/BCF files * merge .. merge VCF/BCF files files from non-overlapping sample sets * mpileup .. multi-way pileup producing genotype likelihoods * norm .. normalize indels * plugin .. run user-defined plugin * polysomy .. detect contaminations and whole-chromosome aberrations * query .. transform VCF/BCF into user-defined formats * reheader .. modify VCF/BCF header, change sample names * roh .. identify runs of homo/auto-zygosity * sort .. sort VCF/BCF files * stats .. produce VCF/BCF stats (former vcfcheck) * view .. subset, filter and convert VCF and BCF files LIST OF SCRIPTS Some helper scripts are bundled with the bcftools code. * gff2gff .. converts a GFF file to the format required by bcftools csq * plot-vcfstats .. plots the output of bcftools stats * roh-viz .. creates HTML/JavaScript visualization of bcftools roh COMMANDS AND OPTIONS Common Options The following options are common to many bcftools commands. See usage for specific commands to see if they apply. FILE Files can be both VCF or BCF, uncompressed or BGZF-compressed. The file "-" is interpreted as standard input. Some tools may require tabix- or CSI-indexed files. -c, --collapse snps|indels|both|all|some|none|id Controls how to treat records with duplicate positions and defines compatible records across multiple input files. Here by "compatible" we mean records which should be considered as identical by the tools. For example, when performing line intersections, the desire may be to con- sider as identical all sites with matching positions (bcftools isec -c all), or only sites with matching variant type (bcftools isec -c snps -c indels), or only sites with all alleles identical (bcftools isec -c none). none only records with identical REF and ALT alleles are compatible some only records where some subset of ALT alleles match are compatible all all records are compatible, regardless of whether the ALT alleles match or not. In the case of records with the same position, only the first will be considered and appear on output. snps any SNP records are compatible, regardless of whether the ALT alle- les match or not. For duplicate positions, only the first SNP record will be considered and appear on output. indels all indel records are compatible, regardless of whether the REF and ALT alleles match or not. For duplicate positions, only the first indel record will be considered and appear on output. both abbreviation of "-c indels -c snps" id only records with identical ID column are compatible. -f, --apply-filters LIST Skip sites where FILTER column does not contain any of the strings listed in LIST. For example, to include only sites which have no fil- ters set, use -f .,PASS. --no-version Do not append version and command line information to the output VCF header. -o, --output FILE When output consists of a single stream, write it to FILE rather than to standard output, where it is written by default. The file type is determined automatically from the file name suffix and in case a con- flicting -O option is given, the file name suffix takes precedence. -O, --output-type b|u|z|v[0-9] Output compressed BCF (b), uncompressed BCF (u), compressed VCF (z), uncompressed VCF (v). Use the -Ou option when piping between bcftools subcommands to speed up performance by removing unnecessary compres- sion/decompression and VCF<-->BCF conversion. The compression level of the compressed formats (b and z) can be set by by appending a number between 0-9. -r, --regions chr|chr:pos|chr:beg-end|chr:beg-[,...] Comma-separated list of regions, see also -R, --regions-file. Overlap- ping records are matched even when the starting coordinate is outside of the region, unlike the -t/-T options where only the POS coordinate is checked. Note that -r cannot be used in combination with -R. -R, --regions-file FILE Regions can be specified either on command line or in a VCF, BED, or tab-delimited file (the default). The columns of the tab-delimited file can contain either positions (two-column format: CHROM, POS) or inter- vals (three-column format: CHROM, BEG, END), but not both. Positions are 1-based and inclusive. The columns of the tab-delimited BED file are also CHROM, POS and END (trailing columns are ignored), but coordi- nates are 0-based, half-open. To indicate that a file be treated as BED rather than the 1-based tab-delimited file, the file must have the ".bed" or ".bed.gz" suffix (case-insensitive). Uncompressed files are stored in memory, while bgzip-compressed and tabix-indexed region files are streamed. Note that sequence names must match exactly, "chr20" is not the same as "20". Also note that chromosome ordering in FILE will be respected, the VCF will be processed in the order in which chromo- somes first appear in FILE. However, within chromosomes, the VCF will always be processed in ascending genomic coordinate order no matter what order they appear in FILE. Note that overlapping regions in FILE can result in duplicated out of order positions in the output. This op- tion requires indexed VCF/BCF files. Note that -R cannot be used in combination with -r. --regions-overlap pos|record|variant|0|1|2 This option controls how overlapping records are determined: set to pos or 0 if the VCF record has to have POS inside a region (this corre- sponds to the default behavior of -t/-T); set to record or 1 if also overlapping records with POS outside a region should be included (this is the default behavior of -r/-R, and includes indels with POS at the end of a region, which are technically outside the region); or set to variant or 2 to include only true overlapping variation (compare the full VCF representation "TA>T-" vs the true sequence variation "A>-"). -s, --samples [^]LIST Comma-separated list of samples to include or exclude if prefixed with "^." (Note that when multiple samples are to be excluded, the "^" pre- fix is still present only once, e.g. "^SAMPLE1,SAMPLE2".) The sample order is updated to reflect that given on the command line. Note that in general tags such as INFO/AC, INFO/AN, etc are not updated to corre- spond to the subset samples. bcftools view is the exception where some tags will be updated (unless the -I, --no-update option is used; see bcftools view documentation). To use updated tags for the subset in an- other command one can pipe from view into that command. For example: bcftools view -Ou -s sample1,sample2 file.vcf | bcftools query -f %INFO/AC\t%INFO/AN\n -S, --samples-file [^]FILE File of sample names to include or exclude if prefixed with "^". One sample per line. See also the note above for the -s, --samples option. The sample order is updated to reflect that given in the input file. The command bcftools call accepts an optional second column indicating ploidy (0, 1 or 2) or sex (as defined by --ploidy, for example "F" or "M"), for example: sample1 1 sample2 2 sample3 2 or sample1 M sample2 F sample3 F If the second column is not present, the sex "F" is assumed. With bcftools call -C trio, PED file is expected. The program ignores the first column and the last indicates sex (1=male, 2=female), for example: ignored_column daughterA fatherA motherA 2 ignored_column sonB fatherB motherB 1 -t, --targets [^]chr|chr:pos|chr:from-to|chr:from-[,...] Similar as -r, --regions, but the next position is accessed by stream- ing the whole VCF/BCF rather than using the tbi/csi index. Both -r and -t options can be applied simultaneously: -r uses the index to jump to a region and -t discards positions which are not in the targets. Unlike -r, targets can be prefixed with "^" to request logical comple- ment. For example, "^X,Y,MT" indicates that sequences X, Y and MT should be skipped. Yet another difference between the -t/-T and -r/-R is that -r/-R checks for proper overlaps and considers both POS and the end position of an indel, while -t/-T considers the POS coordinate only (by default; see also --regions-overlap and --targets-overlap). Note that -t cannot be used in combination with -T. -T, --targets-file [^]FILE Same -t, --targets, but reads regions from a file. Note that -T cannot be used in combination with -t. With the call -C alleles command, third column of the targets file must be comma-separated list of alleles, starting with the reference allele. Note that the file must be compressed and indexed. Such a file can be easily created from a VCF using: bcftools query -f'%CHROM\t%POS\t%REF,%ALT\n' file.vcf | bgzip -c > als.tsv.gz && tabix -s1 -b2 -e2 als.tsv.gz --targets-overlap pos|record|variant|0|1|2 Same as --regions-overlap but for -t/-T. --threads INT Use multithreading with INT worker threads. The option is currently used only for the compression of the output stream, only when --out- put-type is b or z. Default: 0. -v, --verbosity INT Verbosity level. Values bigger than 3 are passed to the underlying HT- Slib library so that network issues and other problems occurring at the library level can be investigated. -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output files. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Defaults to CSI unless specified otherwise. Can be used only for compressed BCF and VCF out- put. bcftools annotate [OPTIONS] FILE Add or remove annotations. -a, --annotations file Bgzip-compressed and tabix-indexed file with annotations. The file can be VCF, BED, or a tab-delimited file with mandatory columns CHROM, POS (or, alternatively, FROM and TO), optional columns REF and ALT, and ar- bitrary number of annotation columns. BED files are expected to have the ".bed" or ".bed.gz" suffix (case-insensitive), otherwise a tab-de- limited file is assumed. Note that in case of tab-delimited file, the coordinates POS, FROM and TO are one-based and inclusive. When REF and ALT are present, only matching VCF records will be annotated. If the END coordinate is present in the annotation file and given on command line as "-c ~INFO/END", then VCF records will be matched also by the INFO/END coordinate. If ID is present in the annotation file and given as "-c ~ID", then VCF records will be matched also by the ID column. When multiple ALT alleles are present in the annotation file (given as comma-separated list of alleles), at least one must match one of the alleles in the corresponding VCF record. Similarly, at least one alter- nate allele from a multi-allelic VCF record must be present in the an- notation file. Missing values can be added by providing "." in place of actual value and using the missing value modifier with -c, such as ".TAG". Note that flag types, such as "INFO/FLAG", can be annotated by includ- ing a field with the value "1" to set the flag, "0" to remove it, or "." to keep existing flags. See also -c, --columns and -h, --header-lines. # Sample annotation file with columns CHROM, POS, STRING_TAG, NUMERIC_TAG 1 752566 SomeString 5 1 798959 SomeOtherString 6 -c, --columns list Comma-separated list of columns or tags to carry over from the annota- tion file (see also -a, --annotations). If the annotation file is not a VCF/BCF, list describes the columns of the annotation file and must include CHROM, POS (or, alternatively, FROM,TO or BEG,END), and optionally REF and ALT. Unused columns which should be ignored can be indicated by "-". If the annotation file is a VCF/BCF, only the edited columns/tags must be present (i.e. leave out CHROM, POS, REF, ALT) and their order does not matter. The columns ID, QUAL, FILTER, INFO and FORMAT can be edited, where INFO tags can be written both as "INFO/TAG" or simply "TAG", and FORMAT tags can be written as "FORMAT/TAG" or "FMT/TAG". The imported VCF annotations can be renamed as "DST_TAG:=SRC_TAG" or "FMT/DST_TAG:=FMT/SRC_TAG". To carry over all INFO annotations, use "INFO". To add all INFO annota- tions except "TAG", use "^INFO/TAG". By default, existing values are replaced. By default, existing tags are overwritten unless the source value is a missing value (i.e. "."). If also missing values should be carried over (and overwrite existing tags), use ".TAG" instead of "TAG". To add an- notations without overwriting existing values (that is, to add tags that are absent or to add values to existing tags with missing values), use "+TAG" instead of "TAG". These can be combined, for example ".+TAG" can be used to add TAG even if the source value is missing but only if TAG does not exist in the target file; existing tags will not be over- written. To append to existing values (rather than replacing or leaving untouched), use "=TAG" (instead of "TAG" or "+TAG"). To replace only existing values without modifying missing annotations, use "-TAG". As a special case of this, if position needs to be replaced, mark the column with the new coordinate as "-POS". (Note that in previous releases this used to be "~POS", now deprecated.) To match the record also by ID or INFO/END, in addition to REF and ALT, use "~ID" or "~INFO/END". Note that this works only for ID and POS, for other fields see the description of -i below. If the annotation file is not a VCF/BCF, all new annotations must be defined via -h, --header-lines. See also the -l, --merge-logic option. Summary of -c, --columns: CHROM,POS,TAG .. match by chromosome and position, transfer annotation from TAG CHROM,POS,-,TAG .. same as above, but ignore the third column of the annotation file CHROM,BEG,END,TAG .. match by region (BEG,END are synonymous to FROM,TO) CHROM,POS,REF,ALT .. match by CHROM, POS, REF and ALT DST_TAG:=SRC_TAG .. transfer the SRC_TAG using the new name DST_TAG INFO .. transfer all INFO annotations ^INFO/TAG .. transfer all INFO annotations except "TAG" TAG .. add or overwrite existing target value if source is not "." and skip otherwise +TAG .. add or overwrite existing target value only if it is "." .TAG .. add or overwrite existing target value even if source is "." .+TAG .. add new but never overwrite existing tag, regardless of its value; can transfer "." if target does not exist -TAG .. overwrite existing value, never add new if target does not exist =TAG .. do not overwrite but append value to existing tags ~FIELD .. use this column to match lines with -i/-e expression (see the description of -i below) ~ID .. in addition to CHROM,POS,REF,ALT match by also ID ~INFO/END .. in addition to CHROM,POS,REF,ALT match by also INFO/END -C, --columns-file file Read the list of columns from a file (normally given via the -c, --columns option). "-" to skip a column of the annotation file. One column name per row, an additional space- or tab-separated field can be present to indicate the merge logic (normally given via the -l, --merge-logic option). This is useful when many annotations are added at once. -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS and the extension described in -i, --include below. --force continue even when parsing errors, such as undefined tags, are encoun- tered. Note this can be an unsafe operation and can result in corrupted BCF files. If this option is used, make sure to sanity check the result thoroughly. -h, --header-lines file Lines to append to the VCF header, see also -c, --columns and -a, --an- notations. For example: ##INFO=<ID=NUMERIC_TAG,Number=1,Type=Integer,Description="Example header line"> ##INFO=<ID=STRING_TAG,Number=1,Type=String,Description="Yet another header line"> -I, --set-id [+]FORMAT assign ID on the fly. The format is the same as in the query command (see below). By default all existing IDs are replaced. If the format string is preceded by "+", only missing IDs will be set. For example, one can use bcftools annotate --set-id +'%CHROM\_%POS\_%REF\_%FIRST_ALT' file.vcf -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. Additionally, the command bcftools annotate supports expressions up- dated from the annotation file dynamically for each record: # The field 'STR' from the -a file is required to match INFO/TAG in VCF. In the first example # the alleles REF,ALT must match, in the second example they are ignored. The option -k is required # to output also records that are not annotated. The third example shows the same concept with # a numerical expression. bcftools annotate -a annots.tsv.gz -c CHROM,POS,REF,ALT,SCORE,~STR -i'TAG={STR}' -k input.vcf bcftools annotate -a annots.tsv.gz -c CHROM,POS,-,-,SCORE,~STR -i'TAG={STR}' -k input.vcf bcftools annotate -a annots.tsv.gz -c CHROM,POS,-,-,SCORE,~INT -i'TAG>{INT}' -k input.vcf -k, --keep-sites keep sites which do not pass -i and -e expressions instead of discard- ing them -l, --merge-logic tag:first|append|append-miss- ing|unique|sum|avg|min|max[,...] When multiple regions overlap a single record, this option defines how to treat multiple annotation values when setting tag in the destination file: use the first encountered value ignoring the rest (first); append allowing duplicates (append); append even if the appended value is missing, i.e. is a dot (append-missing); append discarding duplicate values (unique); sum the values (sum, numeric fields only); average the values (avg); use the minimum value (min) or the maximum (max). + Note that this option is intended for use with BED or TAB-delimited annota- tion files only. Moreover, it is effective only when either REF and ALT or BEG and END --columns are present . + Multiple rules can be given either as a comma-separated list or giving the option multiple times. This is an experimental feature. -m, --mark-sites TAG annotate sites which are present ("+") or absent ("-") in the -a file with a new INFO/TAG flag --min-overlap ANN:'VCF' minimum overlap required as a fraction of the variant in the annotation -a file (ANN), in the target VCF file (:VCF), or both for reciprocal overlap (ANN:VCF). By default overlaps of arbitrary length are suffi- cient. The option can be used only with the tab-delimited annotation -a file and with BEG and END columns present. --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options --pair-logic snps|indels|both|all|some|exact|id Controls how to match records from the annotation file to the target VCF. Effective only when -a is a VCF or BCF. The option replaces the former uninuitive --collapse. See Common Options for more. -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options --rename-annots file rename annotations according to the map in file, with "old_name new_name\n" pairs separated by whitespaces, each on a separate line. The old name must be prefixed with the annotation type: INFO, FORMAT, or FILTER. --rename-chrs file rename chromosomes according to the map in file, with "old_name new_name\n" pairs separated by whitespaces, each on a separate line. -s, --samples [^]LIST subset of samples to annotate, see also Common Options -S, --samples-file FILE subset of samples to annotate. If the samples are named differently in the target VCF and the -a, --annotations VCF, the name mapping can be given as "src_name dst_name\n", separated by whitespaces, each pair on a separate line. --single-overlaps use this option to keep memory requirements low with very large annota- tion files. Note, however, that this comes at a cost, only single over- lapping intervals are considered in this mode. This was the default mode until the commit af6f0c9 (Feb 24 2019). --threads INT see Common Options -x, --remove list List of annotations to remove. Use "FILTER" to remove all filters or "FILTER/SomeFilter" to remove a specific filter. Similarly, "INFO" can be used to remove all INFO tags and "FORMAT" to remove all FORMAT tags except GT. To remove all INFO tags except "FOO" and "BAR", use "^INFO/FOO,INFO/BAR" (and similarly for FORMAT and FILTER). "INFO" can be abbreviated to "INF" and "FORMAT" to "FMT". -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Examples: # Remove three fields bcftools annotate -x ID,INFO/DP,FORMAT/DP file.vcf.gz # Remove all INFO fields and all FORMAT fields except for GT and PL bcftools annotate -x INFO,^FORMAT/GT,FORMAT/PL file.vcf # Add ID, QUAL and INFO/TAG, not replacing TAG if already present bcftools annotate -a src.bcf -c ID,QUAL,+TAG dst.bcf # Carry over all INFO and FORMAT annotations except FORMAT/GT bcftools annotate -a src.bcf -c INFO,^FORMAT/GT dst.bcf # Annotate from a tab-delimited file with six columns (the fifth is ignored), # first indexing with tabix. The coordinates are 1-based. tabix -s1 -b2 -e2 annots.tab.gz bcftools annotate -a annots.tab.gz -h annots.hdr -c CHROM,POS,REF,ALT,-,TAG file.vcf # Annotate from a tab-delimited file with regions (1-based coordinates, inclusive) tabix -s1 -b2 -e3 annots.tab.gz bcftools annotate -a annots.tab.gz -h annots.hdr -c CHROM,FROM,TO,TAG input.vcf # Annotate from a bed file (0-based coordinates, half-closed, half-open intervals) bcftools annotate -a annots.bed.gz -h annots.hdr -c CHROM,FROM,TO,TAG input.vcf # Transfer the INFO/END tag, matching by POS,REF,ALT and ID. This example assumes # that INFO/END is already present in the VCF header. bcftools annotate -a annots.tab.gz -c CHROM,POS,~ID,REF,ALT,INFO/END input.vcf # For (many) more examples see http://samtools.github.io/bcftools/howtos/annotate.html bcftools call [OPTIONS] FILE This command replaces the former bcftools view caller. Some of the original functionality has been temporarily lost in the process of transition under htslib <http://github.com/samtools/htslib>, but will be added back on popu- lar demand. The original calling model can be invoked with the -c option. File format options: --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options --ploidy ASSEMBLY[?] predefined ploidy, use list (or any other unused word) to print a list of all predefined assemblies. Append a question mark to print the ac- tual definition. See also --ploidy-file. --ploidy-file FILE ploidy definition given as a space/tab-delimited list of CHROM, FROM, TO, SEX, PLOIDY. The SEX codes are arbitrary and correspond to the ones used by --samples-file. The default ploidy can be given using the starred records (see below), unlisted regions have ploidy 2. The de- fault ploidy definition is X 1 60000 M 1 X 2699521 154931043 M 1 Y 1 59373566 M 1 Y 1 59373566 F 0 MT 1 16569 M 1 MT 1 16569 F 1 * * * M 2 * * * F 2 -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST see Common Options -S, --samples-file FILE see Common Options -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options --threads INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Input/output options: -A, --keep-alts output all alternate alleles present in the alignments even if they do not appear in any of the genotypes -*, --keep-unseen-allele keep the unobserved allele <*> or <NON_REF>, useful mainly for gVCF output -f, --format-fields list comma-separated list of FORMAT fields to output for each sample. Cur- rently GQ and GP fields are supported. For convenience, the fields can be given as lower case letters. Prefixed with "^" indicates a request for tag removal of auxiliary tags useful only for calling. -F, --prior-freqs AN,AC take advantage of prior knowledge of population allele frequencies. The workflow looks like this: # Extract AN,AC values from an existing VCF, such 1000Genomes bcftools query -f'%CHROM\t%POS\t%REF\t%ALT\t%AN\t%AC\n' 1000Genomes.bcf | bgzip -c > AFs.tab.gz # If the tags AN,AC are not already present, use the +fill-tags plugin bcftools +fill-tags 1000Genomes.bcf | bcftools query -f'%CHROM\t%POS\t%REF\t%ALT\t%AN\t%AC\n' | bgzip -c > AFs.tab.gz tabix -s1 -b2 -e2 AFs.tab.gz # Create a VCF header description, here we name the tags REF_AN,REF_AC cat AFs.hdr ##INFO=<ID=REF_AN,Number=1,Type=Integer,Description="Total number of alleles in reference genotypes"> ##INFO=<ID=REF_AC,Number=A,Type=Integer,Description="Allele count in reference genotypes for each ALT allele"> # Now before calling, stream the raw mpileup output through `bcftools annotate` to add the frequencies bcftools mpileup [...] -Ou | bcftools annotate -a AFs.tab.gz -h AFs.hdr -c CHROM,POS,REF,ALT,REF_AN,REF_AC -Ou | bcftools call -mv -F REF_AN,REF_AC [...] -G, --group-samples FILE|- by default, all samples are assumed to come from a single population. This option groups samples into populations and apply the HWE assump- tion within but not across the populations. FILE is a tab-delimited text file with sample names in the first column and group names in the second column. If - is given instead, no HWE assumption is made at all and single-sample calling is performed. (Note that in low coverage data this inflates the rate of false positives.) The -G option requires the presence of per-sample FORMAT/QS or FORMAT/AD tag generated with bcftools mpileup -a QS (or -a AD). -g, --gvcf INT[,...] output gVCF blocks of homozygous REF calls, with depth (DP) ranges specified by the list of integers. For example, passing 5,15 will group sites into two types of gVCF blocks, the first with minimum per-sample DP from the interval [5,15) and the latter with minimum depth 15 or more. In this example, sites with minimum per-sample depth less than 5 will be printed as separate records, outside of gVCF blocks. -i, --insert-missed INT output also sites missed by mpileup but present in -T, --targets-file. -M, --keep-masked-ref output sites where REF allele is N -V, --skip-variants snps|indels skip indel/SNP sites -v, --variants-only output variant sites only Consensus/variant calling options: -c, --consensus-caller the original samtools/bcftools calling method (conflicts with -m) -C, --constrain alleles|trio alleles call genotypes given alleles. See also -T, --targets-file. trio call genotypes given the father-mother-child constraint. See also -s, --samples and -n, --novel-rate. -m, --multiallelic-caller alternative model for multiallelic and rare-variant calling designed to overcome known limitations in -c calling model (conflicts with -c) -n, --novel-rate float[,...] likelihood of novel mutation for constrained -C trio calling. The trio genotype calling maximizes likelihood of a particular combination of genotypes for father, mother and the child P(F=i,M=j,C=k) = P(uncon- strained) * Pn + P(constrained) * (1-Pn). By providing three values, the mutation rate Pn is set explicitly for SNPs, deletions and inser- tions, respectively. If two values are given, the first is interpreted as the mutation rate of SNPs and the second is used to calculate the mutation rate of indels according to their length as Pn=float*exp(-a-b*len), where a=22.8689, b=0.2994 for insertions and a=21.9313, b=0.2856 for deletions [pubmed:23975140]. If only one value is given, the same mutation rate Pn is used for SNPs and indels. -p, --pval-threshold float with -c, accept variant if P(ref|D) < float. -P, --prior float expected substitution rate, or 0 to disable the prior. Only with -m. -t, --targets file|chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -X, --chromosome-X haploid output for male samples (requires PED file with -s) -Y, --chromosome-Y haploid output for males and skips females (requires PED file with -s) bcftools cnv [OPTIONS] FILE Copy number variation caller, requires a VCF annotated with the Illumina's B-allele frequency (BAF) and Log R Ratio intensity (LRR) values. The HMM considers the following copy number states: CN 2 (normal), 1 (single-copy loss), 0 (complete loss), 3 (single-copy gain). General Options: -c, --control-sample string optional control sample name. If given, pairwise calling is performed and the -P option can be used -f, --AF-file file read allele frequencies from a tab-delimited file with the columns CHR,POS,REF,ALT,AF -o, --output-dir path output directory -p, --plot-threshold float call matplotlib to produce plots for chromosomes with quality at least float, useful for visual inspection of the calls. With -p 0, plots for all chromosomes will be generated. If not given, a matplotlib script will be created but not called. -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --query-sample string query sample name -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options -v, --verbosity INT see Common Options HMM Options: -a, --aberrant float[,float] fraction of aberrant cells in query and control. The hallmark of dupli- cations and contaminations is the BAF value of heterozygous markers which is dependent on the fraction of aberrant cells. Sensitivity to smaller fractions of cells can be increased by setting -a to a lower value. Note however, that this comes at the cost of increased false discovery rate. -b, --BAF-weight float relative contribution from BAF -d, --BAF-dev float[,float] expected BAF deviation in query and control, i.e. the noise observed in the data. -e, --err-prob float uniform error probability -l, --LRR-weight float relative contribution from LRR. With noisy data, this option can have big effect on the number of calls produced. In truly random noise (such as in simulated data), the value should be set high (1.0), but in the presence of systematic noise when LRR are not informative, lower values result in cleaner calls (0.2). -L, --LRR-smooth-win int reduce LRR noise by applying moving average given this window size -O, --optimize float iteratively estimate the fraction of aberrant cells, down to the given fraction. Lowering this value from the default 1.0 to say, 0.3, can help discover more events but also increases noise -P, --same-prob float the prior probability of the query and the control sample being the same. Setting to 0 calls both independently, setting to 1 forces the same copy number state in both. -x, --xy-prob float the HMM probability of transition to another copy number state. In- creasing this values leads to smaller and more frequent calls. bcftools concat [OPTIONS] FILE1 FILE2 [...] Concatenate or combine VCF/BCF files. All source files must have the same sample columns appearing in the same order. Can be used, for example, to concatenate chromosome VCFs into one VCF, or combine a SNP VCF and an indel VCF into one. The input files must be sorted by chr and position. The files must be given in the correct order to produce sorted VCF on output unless the -a, --allow-overlaps option is specified. With the --naive option, the files are concatenated without being recompressed, which is very fast.. -a, --allow-overlaps First coordinate of the next file can precede last record of the cur- rent file. -c, --compact-PS Do not output PS tag at each site, only at the start of a new phase set block. -d, --rm-dups snps|indels|both|all|exact Output duplicate records of specified type present in multiple files only once. Note that records duplicate within one file are not removed with this option, for that use bcftools norm -d instead. In other words, the default behavior of the program is similar to unix "cat" in that when two files contain a record with the same position, that position will appear twice on output. With -d, every line that finds a matching record in another file will be printed only once. Requires -a, --allow-overlaps. -D, --remove-duplicates Alias for -d exact -f, --file-list FILE Read file names from FILE, one file name per line. -l, --ligate Ligate phased VCFs by matching phase at overlapping haplotypes. Note that the option is intended for VCFs with perfect overlap, sites in overlapping regions present in one but missing in the other are dropped. --ligate-force Keep all sites and ligate even non-overlapping chunks and chunks with imperfect overlap --ligate-warn Drop sites in imperfect overlaps --no-version see Common Options -n, --naive Concatenate VCF or BCF files without recompression. This is very fast but requires that all files are of the same type (all VCF or all BCF) and have the same headers. This is because all tags and chromosome names in the BCF body rely on the order of the contig and tag defini- tions in the header. A header check compatibility is performed and the program throws an error if it is not safe to use the option. --naive-force Same as --naive, but header compatibility is not checked. Dangerous, use with caution. -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -q, --min-PQ INT Break phase set if phasing quality is lower than INT -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options. Requires -a, --allow-overlaps. -R, --regions-file FILE see Common Options. Requires -a, --allow-overlaps. --regions-overlap 0|1|2 see Common Options --threads INT see Common Options -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. bcftools consensus [OPTIONS] FILE Create consensus sequence by applying VCF variants to a reference fasta file. By default, the program will apply all ALT variants to the reference fasta to obtain the consensus sequence. Using the --sample (and, option- ally, --haplotype) option will apply genotype (haplotype) calls from FOR- MAT/GT. Note that the program does not act as a primitive variant caller and ignores allelic depth information, such as INFO/AD or FORMAT/AD. For that, consider using the setGT plugin. -a, --absent CHAR replace positions absent from VCF with CHAR -c, --chain FILE write a chain file for liftover -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -f, --fasta-ref FILE reference sequence in fasta format -H, --haplotype N|R|A|I|LR|LA|SR|SA|NpIu choose which allele from the FORMAT/GT field to use (the codes are case-insensitive): N N={1,2,3,...}, the allele index within the genotype, regardless of phasing R the REF allele (in heterozygous genotypes) A the ALT allele (in heterozygous genotypes) I IUPAC code for all genotypes LR, LA the longer allele. If both have the same length, use the REF allele (LR), or the ALT allele (LA) SR, SA the shorter allele. If both have the same length, use the REF al- lele (SR), or the ALT allele (SA) NpIu N={1,2,3,...}, the allele index within genotype for phased geno- types and IUPAC code for unphased genotypes. For example, 1pIu or 2pIu Note that the -H, --haplotype option requires the -s, --samples option, un- less exactly one sample is present in the VCF -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -I, --iupac-codes output variants in the form of IUPAC ambiguity codes determined from FORMAT/GT fields. By default all samples are used and can be subset with -s, --samples and -S, --samples-file. Use -s - to ignore samples and use only the REF and ALT columns. NOTE: prior to version 1.17 the IUPAC codes were determined solely from REF,ALT columns and sample genotypes were not considered. --mark-del CHAR instead of removing sequence, insert character CHAR for deletions --mark-ins uc|lc|CHAR highlight inserted sequence in uppercase (uc), lowercase (lc), or a provided character CHAR, leaving the rest of the sequence as is --mark-snv uc|lc highlight substitutions in uppercase (uc), lowercase (lc), or a pro- vided character CHAR, leaving the rest of the sequence as is -m, --mask FILE BED file or TAB file with regions to be replaced with N (the default) or as specified by the next --mask-with option. See discussion of --re- gions-file in Common Options for file format details. --mask-with CHAR|lc|uc replace sequence from --mask with CHAR, skipping overlapping variants, or change to lowercase (lc) or uppercase (uc) -M, --missing CHAR instead of skipping the missing genotypes, output the character CHAR (e.g. "?") -o, --output FILE write output to a file --regions-overlap 0|1|2 how to treat VCF variants overlapping the target region in the fasta file: see Common Options -s, --samples LIST apply variants of the listed samples. See also the option -I, --iu- pac-codes -S, --samples-file FILE apply variants of the samples listed in the file. See also the option -I, --iupac-codes -v, --verbosity INT see Common Options Examples: # Apply variants present in sample "NA001", output IUPAC codes for hets bcftools consensus -i -s NA001 -f in.fa in.vcf.gz > out.fa # Create consensus for one region. The fasta header lines are then expected # in the form ">chr:from-to". Ignore samples and consider only the REF and ALT columns samtools faidx ref.fa 8:11870-11890 | bcftools consensus -s - in.vcf.gz -o out.fa # For more examples see http://samtools.github.io/bcftools/howtos/consensus-sequence.html Notes: Masking options are applied in the following order 1. mask regions with --mask-with character if --mask is given. All overlapping VCF variants are ignored 2. replace sequence not mentioned in the VCF with the requested char- acter if --absent is given 3. finally apply --mark-del, --mark-ins, --mark-snv masks bcftools convert [OPTIONS] FILE VCF input options: -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file FILE see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST see Common Options -S, --samples-file FILE see Common Options -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. General options: --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options --threads INT see Common Options -v, --verbosity INT see Common Options GEN/SAMPLE conversion: -G, --gensample2vcf prefix or gen-file,sample-file convert IMPUTE2 output to VCF. One of the ID columns ("SNP ID" or "rsID" in https://www.cog-genomics.org/plink/2.0/formats#gen) must be of the form "CHROM:POS_REF_ALT" to detect possible strand swaps. When the --vcf-ids option is given, the other column (autodetected) is used to fill the ID column of the VCF. See also -g and --3N6 options. -g, --gensample prefix or gen-file,sample-file convert from VCF to gen/sample format used by IMPUTE2 and SHAPEIT. The columns of .gen file format are ID1,ID2,POS,A,B followed by three geno- type probabilities P(AA), P(AB), P(BB) for each sample. In order to prevent strand swaps, the program uses IDs of the form "CHROM:POS_REF_ALT". When the --vcf-ids option is given, the second column is set to match the ID column of the VCF. See also -G and --3N6 options. The file .gen and .sample file format are: .gen (with --3N6 --vcf-ids) --------------------------- chr1 1:111485207_G_A rsID1 111485207 G A 0 1 0 0 1 0 chr1 1:111494194_C_T rsID2 111494194 C T 0 1 0 0 0 1 .gen (with --vcf-ids) --------------------------- 1:111485207_G_A rsID1 111485207 G A 0 1 0 0 1 0 1:111494194_C_T rsID2 111494194 C T 0 1 0 0 0 1 .gen (the default) ------------------------------ 1:111485207_G_A 1:111485207_G_A 111485207 G A 0 1 0 0 1 0 1:111494194_C_T 1:111494194_C_T 111494194 C T 0 1 0 0 0 1 .sample ------- ID_1 ID_2 missing 0 0 0 sample1 sample1 0 sample2 sample2 0 --3N6 Expect/Create files in the 3*N+6 column format. This is the new .gen file format with the first column containing the chromosome name, see https://www.cog-genomics.org/plink/2.0/formats#gen --tag STRING tag to take values for .gen file: GT,PL,GL,GP --sex FILE output sex column in the sample file. The FILE format is MaleSample M FemaleSample F --vcf-ids output VCF IDs in the second column instead of CHROM:POS_REF_ALT gVCF conversion: --gvcf2vcf convert gVCF to VCF, expanding REF blocks into sites. Note that the -i and -e options work differently with this switch. In this situation the filtering expressions define which sites should be expanded and which sites should be left unmodified, but all sites are printed on output. In order to drop sites, stream first through bcftools view. -f, --fasta-ref file reference sequence in fasta format. Must be indexed with samtools faidx HAP/SAMPLE conversion: --hapsample2vcf prefix or hap-file,sample-file convert from hap/sample format to VCF. The columns of .hap file are similar to .gen file above, but there are only two haplotype columns per sample. Note that the first or the second column of the .hap file is expected to be in the form "CHR:POS_REF_ALT[_END]", with the _END being optional for defining the INFO/END tag when ALT is a symbolic al- lele. For example: .hap (with --vcf-ids) --------------------- 1:111485207_G_A rsID1 111485207 G A 0 1 0 0 1:111495231_A_<DEL>_111495784 rsID3 111495231 A <DEL> 0 0 1 0 .hap (the default) ------------------ 1 1:111485207_G_A 111485207 G A 0 1 0 0 1 1:111495231_A_<DEL>_111495784 111495231 A <DEL> 0 0 1 0 --hapsample prefix or hap-file,sample-file convert from VCF to hap/sample format used by IMPUTE2 and SHAPEIT. The columns of .hap file begin with ID,RSID,POS,REF,ALT. In order to pre- vent strand swaps, the program uses IDs of the form "CHROM:POS_REF_ALT". --haploid2diploid with -h option converts haploid genotypes to homozygous diploid geno- types. For example, the program will print 0 0 instead of the default 0 -. This is useful for programs which do not handle haploid genotypes correctly. --sex FILE output sex column in the sample file. The FILE format is MaleSample M FemaleSample F --vcf-ids the second column of the .hap file holds the VCF ids, the first column is of the form "CHR:POS_REF_ALT[_END]". Without the option, the format follows https://www.cog-genomics.org/plink/2.0/formats#haps with ids (the second column) of the form "CHR:POS_REF_ALT[_END]" HAP/LEGEND/SAMPLE conversion: -H, --haplegendsample2vcf prefix or hap-file,legend-file,sample-file convert from hap/legend/sample format used by IMPUTE2 to VCF. See also -h, --hapslegendsample below. -h, --haplegendsample prefix or hap-file,legend-file,sample-file convert from VCF to hap/legend/sample format used by IMPUTE2 and SHAPEIT. The columns of .legend file ID,POS,REF,ALT. In order to pre- vent strand swaps, the program uses IDs of the form "CHROM:POS_REF_ALT". The .sample file is quite basic at the moment with columns for population, group and sex expected to be edited by the user. For example: .hap ----- 0 1 0 0 1 0 0 1 0 0 0 1 .legend ------- id position a0 a1 1:111485207_G_A 111485207 G A 1:111494194_C_T 111494194 C T .sample ------- sample population group sex sample1 sample1 sample1 2 sample2 sample2 sample2 2 --haploid2diploid with -h option converts haploid genotypes to homozygous diploid geno- types. For example, the program will print 0 0 instead of the default 0 -. This is useful for programs which do not handle haploid genotypes correctly. --sex FILE output sex column in the sample file. The FILE format is MaleSample M FemaleSample F --vcf-ids output VCF IDs instead of "CHROM:POS_REF_ALT". Note that this option can be used with --haplegendsample but not with --haplegendsample2vcf. TSV conversion: --tsv2vcf file convert from TSV (tab-separated values) format (such as generated by 23andMe) to VCF. The input file fields can be tab- or space- delimited -c, --columns list comma-separated list of fields in the input file. In the current ver- sion, the fields CHROM, POS, ID, and AA or REF, ALT are expected and can appear in arbitrary order. Columns which should be ignored in the input file can be indicated by "-". The AA field lists alleles on the forward reference strand, for example "CC" or "CT" for diploid geno- types or "C" for haploid genotypes (sex chromosomes). Insertions and deletions are supported only with REF and ALT but not with AA. Missing data can be indicated with "--" or ".". -f, --fasta-ref file reference sequence in fasta format. Must be indexed with samtools faidx -s, --samples LIST list of sample names. See Common Options -S, --samples-file FILE file of sample names. See Common Options Example: # Convert 23andme results into VCF bcftools convert -c ID,CHROM,POS,AA -s SampleName -f 23andme-ref.fa --tsv2vcf 23andme.txt -o out.vcf.gz # Convert tab-delimited file into a sites-only VCF (no genotypes), in this example first column to be ignored bcftools convert -c -,CHROM,POS,REF,ALT -f ref.fa --tsv2vcf calls.txt -o out.bcf bcftools csq [OPTIONS] FILE Haplotype aware consequence predictor which correctly handles combined variants such as MNPs split over multiple VCF records, SNPs separated by an intron (but adjacent in the spliced transcript) or nearby frame-shifting indels which in combination in fact are not frame-shifting. The output VCF is annotated with INFO/BCSQ and FORMAT/BCSQ tag (config- urable with the -c option). The latter is a bitmask of indexes to INFO/BCSQ, with interleaved haplotypes. See the usage examples below for using the %TBCSQ converter in query for extracting a more human readable form from this bitmask. The construction of the bitmask limits the number of consequences that can be referenced per sample in the FORMAT/BCSQ tags. By default this is 15, but if more are required, see the --ncsq option. Note that the program annotates only records with a functional consequence and intergenic regions will pass through unchanged. The program requires on input a VCF/BCF file, the reference genome in fasta format (--fasta-ref) and genomic features in the GFF3 format downloadable from the Ensembl website (--gff-annot), and outputs an annotated VCF/BCF file. Currently, only Ensembl GFF3 files are supported. By default, the input VCF should be phased. If phase is unknown, or only partially known, the --phase option can be used to indicate how to handle unphased data. Alternatively, haplotype aware calling can be turned off with the --local-csq option. If conflicting (overlapping) variants within one haplotype are detected, a warning will be emitted and predictions will be based on only the first variant in the analysis. Symbolic alleles are not supported. They will remain unannotated in the output VCF and are ignored for the prediction analysis. -B, --trim-protein-seq INT abbreviate protein-changing predictions to maximum of INT aminoacids. For example, instead of writing the whole modified protein sequence with potentially hundreds of aminoacids, with -B 1 only an abbreviated version such as 25E..329>25G..94 will be written. -C, --genetic-code INT|l specify the genetic code table to use, l to print a list of supported tables -c, --custom-tag STRING use this custom tag to store consequences rather than the default BCSQ tag --dump-gff FILE dump the parsed GFF into a gzipped FILE. Intended for debugging pur- poses, shows how is the input GFF viewed by the program. -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -f, --fasta-ref FILE reference sequence in fasta format (required) --force run even if some sanity checks fail. Currently the option enables skip- ping transcripts in malformatted GFFs with incorrect phase -g, --gff-annot FILE GFF3 annotation file (required), such as http://ftp.ensembl.org/pub/current_gff3/homo_sapiens/. The script gff2gff can help with conversion from non-standard GFF formats. An ex- ample of a minimal working GFF file: # The program looks for "CDS", "exon", "three_prime_UTR" and "five_prime_UTR" lines, # looks up their parent transcript (determined from the "Parent=transcript:" attribute), # the gene (determined from the transcript's "Parent=gene:" attribute), and the biotype # (the most interesting is "protein_coding"). # # Empty and commented lines are skipped, the following GFF columns are required # 1. chromosome # 2. IGNORED # 3. type (CDS, exon, three_prime_UTR, five_prime_UTR, gene, transcript, etc.) # 4. start of the feature (1-based) # 5. end of the feature (1-based) # 6. IGNORED # 7. strand (+ or -) # 8. phase (0, 1, 2 or .) # 9. semicolon-separated attributes (see below) # # Attributes required for # gene lines: # - ID=gene:<gene_id> # - biotype=<biotype> # - Name=<gene_name> [optional] # # transcript lines: # - ID=transcript:<transcript_id> # - Parent=gene:<gene_id> # - biotype=<biotype> # # other lines (CDS, exon, five_prime_UTR, three_prime_UTR): # - Parent=transcript:<transcript_id> # # Supported biotypes: # - see the function gff_parse_biotype() in bcftools/csq.c 1 ignored_field gene 21 2148 . - . ID=gene:GeneId;biotype=protein_coding;Name=GeneName 1 ignored_field transcript 21 2148 . - . ID=transcript:TranscriptId;Parent=gene:GeneId;biotype=protein_coding 1 ignored_field three_prime_UTR 21 2054 . - . Parent=transcript:TranscriptId 1 ignored_field exon 21 2148 . - . Parent=transcript:TranscriptId 1 ignored_field CDS 21 2148 . - 1 Parent=transcript:TranscriptId 1 ignored_field five_prime_UTR 210 2148 . - . Parent=transcript:TranscriptId -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -l, --local-csq switch off haplotype-aware calling, run localized predictions consider- ing only one VCF record at a time -n, --ncsq INT maximum number of per-haplotype consequences to consider for each site. The INFO/BCSQ column includes all consequences, but only the first INT will be referenced by the FORMAT/BCSQ fields. The default value is 15 which corresponds to one 32-bit integer per diploid sample, after ac- counting for values reserved by the BCF specification. Note that in- creasing the value leads to increased size of the output BCF. --no-version see Common Options -o, --output FILE see Common Options -O, --output-type t|b|u|z|v[0-9] see Common Options. In addition, a custom tab-delimited plain text out- put can be printed (t). -p, --phase a|m|r|R|s how to handle unphased heterozygous genotypes: a take GTs as is, create haplotypes regardless of phase (0/1 -> 0|1) m merge all GTs into a single haplotype (0/1 -> 1, 1/2 -> 1) r require phased GTs, throw an error on unphased heterozygous GTs R create non-reference haplotypes if possible (0/1 -> 1|1, 1/2 -> 1|2) s skip unphased heterozygous GTs -q, --quiet suppress warning messages -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file FILE see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST samples to include or "-" to apply all variants and ignore samples -S, --samples-file FILE see Common Options -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options --unify-chr-names 0|LIST unify chromosome naming by stripping a prefix in VCF, GFF, fasta, re- spectively. For example, if the LIST is "chr,Chromosome,-", the program will trim the "chr" prefix in the VCF, "Chromosome" in the GFF, and the fasta will be left unchanged. The chromosome names in the output VCF will match that of the input VCF. The default is not to modify the se- quence names. -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Examples: # Basic usage bcftools csq -f hs37d5.fa -g Homo_sapiens.GRCh37.82.gff3.gz in.vcf -Ob -o out.bcf # Extract the translated haplotype consequences. The following TBCSQ variations # are recognised: # %TBCSQ .. print consequences in all haplotypes in separate columns # %TBCSQ{0} .. print the first haplotype only # %TBCSQ{1} .. print the second haplotype only # %TBCSQ{*} .. print a list of unique consequences present in either haplotype bcftools query -f'[%CHROM\t%POS\t%SAMPLE\t%TBCSQ\n]' out.bcf Examples of BCSQ annotation: # Two separate VCF records at positions 2:122106101 and 2:122106102 # change the same codon. This UV-induced C>T dinucleotide mutation # has been annotated fully at the position 2:122106101 with # - consequence type # - gene name # - ensembl transcript ID # - coding strand (+ fwd, - rev) # - amino acid position (in the coding strand orientation) # - list of corresponding VCF variants # The annotation at the second position gives the position of the full # annotation BCSQ=missense|CLASP1|ENST00000545861|-|1174P>1174L|122106101G>A+122106102G>A BCSQ=@122106101 # A frame-restoring combination of two frameshift insertions C>CG and T>TGG BCSQ=@46115084 BCSQ=inframe_insertion|COPZ2|ENST00000006101|-|18AGRGP>18AQAGGP|46115072C>CG+46115084T>TGG # Stop gained variant BCSQ=stop_gained|C2orf83|ENST00000264387|-|141W>141*|228476140C>T # The consequence type of a variant downstream from a stop are prefixed with * BCSQ=*missense|PER3|ENST00000361923|+|1028M>1028T|7890117T>C Supported consequence types 3_prime_utr 5_prime_utr coding_sequence feature_elongation frameshift inframe_altering inframe_deletion inframe_insertion intergenic intron missense non_coding splice_acceptor splice_donor splice_region start_lost start_retained stop_gained stop_lost stop_retained synonymous See also https://ensembl.org/info/genome/variation/prediction/predicted_data.html bcftools filter [OPTIONS] FILE Apply fixed-threshold filters. -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -g, --SnpGap INT[:'indel',mnp,bnd,other,overlap] filter SNPs within INT base pairs of an indel or other other variant type. The following example demonstrates the logic of --SnpGap 3 ap- plied on a deletion and an insertion: The SNPs at positions 1 and 7 are filtered, positions 0 and 8 are not: 0123456789 ref .G.GT..G.. del .A.G-..A.. Here the positions 1 and 6 are filtered, 0 and 7 are not: 0123-456789 ref .G.G-..G.. ins .A.GT..A.. -G, --IndelGap INT filter clusters of indels separated by INT or fewer base pairs allowing only one to pass. The following example demonstrates the logic of --In- delGap 2 applied on a deletion and an insertion: The second indel is filtered: 012345678901 ref .GT.GT..GT.. del .G-.G-..G-.. And similarly here, the second is filtered: 01 23 456 78 ref .A-.A-..A-.. ins .AT.AT..AT.. -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. --mask [^]REGION Soft filter regions, prepepend "^" to negate. Requires -s, --soft-fil- ter. -M, --mask-file [^]FILE Soft filter regions listed in a file, "^" to negate. Requires -s, --soft-filter. --mask-overlap 0|1|2 Same as --regions-overlap but for --mask/--mask-file. See Common Op- tions. [1] -m, --mode [+x] define behaviour at sites with existing FILTER annotations. The default mode replaces existing filters of failed sites with a new FILTER string while leaving sites which pass untouched when non-empty and setting to "PASS" when the FILTER string is absent. The "+" mode appends new FIL- TER strings of failed sites instead of replacing them. The "x" mode re- sets filters of sites which pass to "PASS". Modes "+" and "x" can both be set. --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --soft-filter STRING|+ annotate FILTER column with STRING or, with +, a unique filter name generated by the program ("Filter%d"). Applies to records that do not meet filter expression. -S, --set-GTs .|0 set genotypes of failed samples to missing value (.) or reference al- lele (0) -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options --threads INT see Common Options -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. bcftools gtcheck [OPTIONS] [-g genotypes.vcf.gz] query.vcf.gz Checks sample identity. The program can operate in two modes. If the -g op- tion is given, the identity of samples from query.vcf.gz is checked against the samples in the -g file. Without the -g option, multi-sample cross-check of samples in query.vcf.gz is performed. Note that the interpretation of the discordance score depends on the op- tions provided (specifically -e and -u) and on the available annotations (FORMAT/PL vs FORMAT/GT). The discordance score can be interpreted as the number of mismatching genotypes only if GT-vs-GT matching is performed. --distinctive-sites NUM[,MEM[,DIR]] Find sites that can distinguish between at least NUM sample pairs. If the number is smaller or equal to 1, it is interpreted as the fraction of pairs. The optional MEM string sets the maximum memory used for in-memory sorting and DIR is the temporary directory for external sort- ing. This option requires also --pairs to be given. --dry-run Stop after first record to estimate required time. -e, --exclude [qry|gt]:'EXPRESSION' Exclude sites from query file (qry:) or genotype file (gt:) for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -E, --error-probability INT Interpret genotypes and genotype likelihoods probabilistically. The value of INT represents genotype quality when GT tag is used (e.g. Q=30 represents one error in 1,000 genotypes and Q=40 one error in 10,000 genotypes) and is ignored when PL tag is used (in that case an arbi- trary non-zero integer can be provided). If -E is set to 0, the discordance score can be interpreted as the num- ber of mismatching genotypes, but only in the GT-vs-GT matching mode. See the -u, --use option below for additional notes and caveats. If performance is an issue, set -E 0 for faster run times but less ac- curate results. Note that in previous versions of bcftools (<=1.18), this option used to be a smaller case -e. It changed to make room for the filtering op- tion -e, --exclude to stay consistent across other commands. -g, --genotypes FILE VCF/BCF file with reference genotypes to compare against -H, --homs-only Homozygous genotypes only, useful with low coverage data (requires -g, --genotypes) -i, --include [qry|gt]:'EXPRESSION' Include sites from query file (qry:) or genotype file (gt:) for which EXPRESSION is true. For valid expressions see EXPRESSIONS. --keep-refs Include monoallelic sites in comparison, by default they are excluded --n-matches INT Print only top INT matches for each sample, 0 for unlimited. Use nega- tive value to sort by HWE probability rather than the number of discor- dant sites. Note that average score is used to determine the top matches, not absolute values. --no-HWE-prob Disable calculation of HWE probability to reduce memory requirements with comparisons between very large number of sample pairs. -o, --output FILE Write to FILE rather than to standard output, where it is written by default. -O, --output-type t|z Write a plain (t) or compressed (z) text tab-delimited output. -p, --pairs LIST A comma-separated list of sample pairs to compare. When the -g option is given, the first sample must be from the query file, the second from the -g file, third from the query file etc (qry,gt[,qry,gt..]). Without the -g option, the pairs are created the same way but both samples are from the query file (qry,qry[,qry,qry..]) -P, --pairs-file FILE A file with tab-delimited sample pairs to compare. The first sample in the pair must come from the query file, the second from the genotypes file when -g is given -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] Restrict to comma-separated list of regions, see Common Options *-R, --regions-file' FILE Restrict to regions listed in a file, see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples [qry|gt]:'LIST': List of query samples or -g samples. If nei- ther -s nor -S are given, all possible sample pair combinations are com- pared -S, --samples-file [qry|gt]:'FILE' File with the query or -g samples to compare. If neither -s nor -S are given, all possible sample pair combina- tions are compared -t, --targets file see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options -u, --use TAG1[,TAG2] specifies which tag to use in the query file (TAG1) and the -g (TAG2) file. By default, the PL tag is used in the query file and, when avail- able, the GT tags in the -g file. Note that when the requested tag is not available, the program will at- tempt to use the other tag. The output includes the number of sites that were matched by the four possible modes (for example GT-vs-GT or GT-vs-PL). -v, --verbosity INT see Common Options Examples: # Check discordance of all samples from B against all samples in A bcftools gtcheck -g A.bcf B.bcf # Limit comparisons to the given list of samples bcftools gtcheck -s gt:a1,a2,a3 -s qry:b1,b2 -g A.bcf B.bcf # Compare only two pairs a1,b1 and a1,b2 bcftools gtcheck -p a1,b1,a1,b2 -g A.bcf B.bcf bcftools head [OPTIONS] [FILE] By default, prints all headers from the specified input file to standard output in VCF format. The input file may be in VCF or BCF format; if no FILE is specified, standard input will be read. With appropriate options, only some of the headers and/or additionally some of the variant records will be printed. The bcftools head command outputs VCF headers almost exactly as they appear in the input file: it may add a ##FILTER=<ID=PASS> header if not already present, but it never adds version or command line information itself. Options: -h, --header INT Display only the first INT header lines. By default, all header lines are displayed. -n, --records INT Also display the first INT variant records. By default, no variant records are displayed. -s, --samples INT Display the first INT variant records including the last #CHROM header line with samples. Running with -s 0 alone outputs the #CHROM header line only. Note that the list of samples, with each sample per line, can be obtained with bcftools query using the option -l, --list-sam- ples. -v, --verbosity INT see Common Options bcftools index [OPTIONS] in.bcf|in.vcf.gz Creates index for bgzip compressed VCF/BCF files for random access. CSI (coordinate-sorted index) is created by default. The CSI format supports indexing of chromosomes up to length 2^31. TBI (tabix index) index files, which support chromosome lengths up to 2^29, can be created by using the -t/--tbi option or using the tabix program packaged with htslib. When load- ing an index file, bcftools will try the CSI first and then the TBI. Indexing options: -c, --csi generate CSI-format index for VCF/BCF files [default] -f, --force overwrite index if it already exists -m, --min-shift INT set minimal interval size for CSI indices to 2^INT; default: 14 -o, --output FILE output file name. If not set, then the index will be created using the input file name plus a .csi or .tbi extension -t, --tbi generate TBI-format index for VCF files --threads INT see Common Options -v, --verbosity INT see Common Options Stats options: -a, --all Used in conjunction with -s, --stats, print per contig stats for all contigs, even those with zero records and those for which no stats are recorded in the index file (shown as .). -n, --nrecords print the number of records based on the CSI or TBI index files -s, --stats Print per contig stats based on the CSI or TBI index files. Output for- mat is three tab-delimited columns listing the contig name, contig length (. if unknown) and number of records for the contig. Contigs with zero records are not printed by default. bcftools isec [OPTIONS] A.vcf.gz B.vcf.gz [...] Creates intersections, unions and complements of VCF files. Depending on the options, the program can output records from one (or more) files which have (or do not have) corresponding records with the same position in the other files. -c, --collapse snps|indels|both|all|some|none|id see Common Options -C, --complement output positions present only in the first file but missing in the oth- ers -e, --exclude -|EXPRESSION exclude sites for which EXPRESSION is true. If -e (or -i) appears only once, the same filtering expression will be applied to all input files. Otherwise, -e or -i must be given for each input file. To indicate that no filtering should be performed on a file, use "-" in place of EXPRES- SION, as shown in the example below. For valid expressions see EXPRES- SIONS. -f, --apply-filters LIST see Common Options -i, --include EXPRESSION include only sites for which EXPRESSION is true. See discussion of -e, --exclude above. -f, --file-list FILE Read file names from FILE, one file name per line. -n, --nfiles [+-=]INT|~BITMAP output positions present in this many (=), this many or more (+), this many or fewer (-), or the exact same (~) files -o, --output FILE see Common Options. When several files are being output, their names are controlled via -p instead. -O, --output-type b|u|z|v[0-9] see Common Options -p, --prefix DIR if given, subset each of the input files accordingly. See also -w. -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options -v, --verbosity INT see Common Options -w, --write LIST comma-separated list of input files to output given as 1-based indices. With -p and no -w, all files are written. -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and defaults to tbi for vcf.gz and csi for bcf. This is done automatically with the -p option if the output format is compressed. Examples: Create intersection and complements of two sets saving the output in dir/* bcftools isec -p dir A.vcf.gz B.vcf.gz Filter sites in A (require INFO/MAF>=0.01) and B (require INFO/dbSNP) but not in C, and create an intersection, including only sites which appear in at least two of the files after filters have been applied bcftools isec -e'MAF<0.01' -i'dbSNP=1' -e- A.vcf.gz B.vcf.gz C.vcf.gz -n +2 -p dir Extract and write records from A shared by both A and B using exact allele match bcftools isec -p dir -n=2 -w1 A.vcf.gz B.vcf.gz Extract records private to A or B comparing by position only bcftools isec -p dir -n-1 -c all A.vcf.gz B.vcf.gz Print a list of records which are present in A and B but not in C and D bcftools isec -n~1100 -c all A.vcf.gz B.vcf.gz C.vcf.gz D.vcf.gz bcftools merge [OPTIONS] A.vcf.gz B.vcf.gz [...] Merge multiple VCF/BCF files from non-overlapping sample sets to create one multi-sample file. For example, when merging file A.vcf.gz containing sam- ples S1, S2 and S3 and file B.vcf.gz containing samples S3 and S4, the out- put file will contain five samples named S1, S2, S3, 2:S3 and S4. Note that it is responsibility of the user to ensure that the sample names are unique across all files. If they are not, the program will exit with an error unless the option --force-samples is given. The sample names can be also given explicitly using the --print-header and --use-header options. Note that only records from different files can be merged, never from the same file. For "vertical" merge take a look at bcftools concat or bcftools norm -m instead. --force-no-index synonymous to --no-index --force-samples if the merged files contain duplicate samples names, proceed anyway. Duplicate sample names will be resolved by prepending the index of the file as it appeared on the command line to the conflicting sample name (see 2:S3 in the above example). --force-single run even if only one file is given on input --print-header print only merged header and exit --use-header FILE use the VCF header in the provided text FILE -0 --missing-to-ref assume genotypes at missing sites are 0/0 -f, --apply-filters LIST see Common Options -F, --filter-logic x|+ Set the output record to PASS if any of the inputs is PASS (x), or ap- ply all filters (+), which is the default. -g, --gvcf -|FILE merge gVCF blocks, INFO/END tag is expected. If the reference fasta file FILE is not given and the dash (-) is given, unknown reference bases generated at gVCF block splits will be substituted with N's. The --gvcf option uses the following default INFO rules: -i QS:sum,MinDP:min,I16:sum,IDV:max,IMF:max and the following missing rules: -M PL:max,AD:0. -i, --info-rules -|TAG:METHOD[,...] Rules for merging INFO fields (scalars or vectors) or - to disable the default rules. METHOD is one of sum, avg, min, max, join. Default is DP:sum,DP4:sum if these fields exist in the input files. Fields with no specified rule will take the value from the first input file. The merged QUAL value is currently set to the maximum. This behaviour is not user controllable at the moment. -l, --file-list FILE Read file names from FILE, one file name per line. -L, --local-alleles INT Sites with many alternate alleles can require extremely large storage space which can exceed the 2GB size limit representable by BCF. This is caused by Number=G tags (such as FORMAT/PL) which store a value for each combination of reference and alternate alleles. The -L, --lo- cal-alleles option allows replacement of such tags with a localized tag (FORMAT/LPL) which only includes a subset of alternate alleles relevant for that sample. A new FORMAT/LAA tag is added which lists 1-based in- dices of the alternate alleles relevant (local) for the current sample. The number INT gives the maximum number of alternate alleles that can be included in the PL tag. The default value is 0 which disables the feature and outputs values for all alternate alleles. -m, --merge snps|indels|both|snp-ins-del|all|none|id[,*] The option controls what types of multiallelic records can be created. If single asterisk * is appended, the unobserved allele <*> or <NON_REF> will be removed at variant sites; if two asterisks ** are ap- pended, the unobserved allele will be removed all sites. -m none .. no new multiallelics, output multiple records instead -m snps .. allow multiallelic SNP records -m indels .. allow multiallelic indel records -m both .. both SNP and indel records can be multiallelic -m both,* .. same as above but remove <*> (or <NON_REF>) from variant sites -m both,** .. same as above but remove <*> (or <NON_REF>) at all sites -m all .. SNP records can be merged with indel records -m snp-ins-del .. allow multiallelic SNVs, insertions, deletions, but don't mix them -m id .. merge by ID -M, --missing-rules -|TAG:METHOD[,...] Rules for merging vector tags at multiallelic sites. When input files have different alternate alleles, vector fields pertaining to unob- served alleles are set to missing (.) by default. The METHOD is one of . (the default, use missing values), NUMBER (use a constant value, e.g. 0), max (the maximum value observed for other alleles in the sample). When --gvcf option is set, the rule -M PL:max,AD:0 is implied. This can be overridden with providing -M - or -M PL:.,AD:.. Note that if the un- observed allele is explicitly present as <*> or <NON_REF>, then its corresponding value will be used regardless of -M settings. --no-index the option allows files to be merged without indexing them first. In order for this option to work, the user must ensure that the input files have chromosomes in the same order and consistent with the order of sequences in the VCF header. --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options --threads INT see Common Options -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. bcftools mpileup [OPTIONS] -f ref.fa in.bam [in2.bam [...]] Generate VCF or BCF containing genotype likelihoods for one or multiple alignment (BAM or CRAM) files. This is based on the original samtools mpileup command (with the -v or -g options) producing genotype likelihoods in VCF or BCF format, but not the textual pileup output. The mpileup com- mand was transferred to bcftools in order to avoid errors resulting from use of incompatible versions of samtools and bcftools when using in the mpileup+bcftools call pipeline. Individuals are identified from the SM tags in the @RG header lines. Multi- ple individuals can be pooled in one alignment file, also one individual can be separated into multiple files. If sample identifiers are absent, each input file is regarded as one sample. Note that there are two orthogonal ways to specify locations in the input file; via -r region and -t positions. The former uses (and requires) an in- dex to do random access while the latter streams through the file contents filtering out the specified regions, requiring no index. The two may be used in conjunction. For example a BED file containing locations of genes in chromosome 20 could be specified using -r 20 -t chr20.bed, meaning that the index is used to find chromosome 20 and then it is filtered for the re- gions listed in the BED file. Also note that the -r option can be much slower than -t with many regions and can require more memory when multiple regions and many alignment files are processed. Input options -6, --illumina1.3+ Assume the quality is in the Illumina 1.3+ encoding. -A, --count-orphans Include anomalous read pairs in variant calling, i.e. reads with flag PAIRED but not PROPER_PAIR set. By default such reads are discarded. -b, --bam-list FILE List of input alignment files, one file per line [null] -B, --no-BAQ Disable probabilistic realignment for the computation of base alignment quality (BAQ). BAQ is the Phred-scaled probability of a read base being misaligned. Applying this option greatly helps to reduce false SNPs caused by misalignments. -C, --adjust-MQ INT Coefficient for downgrading mapping quality for reads containing exces- sive mismatches. Mismatches are counted as a proportion of the number of aligned bases ("M", "X" or "=" CIGAR operations), along with their quality, to derive an upper-bound of the mapping quality. Original map- ping qualities lower than this are left intact, while higher ones are capped at the new adjusted score. The exact formula is complex and likely tuned to specific instruments and specific alignment tools, so this option is disabled by default (indicated as having a zero value). Variables in the formulae and their meaning are defined below. Variable Meaning / formula M The number of matching CIGAR bases (operation "M", "X" or "="). X The number of substitutions with quality >= 13. SubQ The summed quality of substitution bases included in X, capped at a maximum of quality 33 per mismatching base. ClipQ The summed quality of soft-clipped or hard-clipped bases. This has no minimum or maximum quality threshold per base. For hard-clipped bases the per-base quality is taken as 13. T SubQ - 10 * log10(M^X / X!) + ClipQ/5 Cap MAX(0, INT * sqrt((INT - T) / INT)) Some notes on the impact of this. * As the number of mismatches increases, the mapping quality cap re- duces, eventually resulting in discarded alignments. * High quality mismatches reduces the cap faster than low quality mismatches. * The starting INT value also acts as a hard cap on mapping quality, even when zero mismatches are observed. * Indels have no impact on the mapping quality. The intent of this option is to work around aligners that compute a mapping quality using a local alignment without having any regard to the degree of clipping required or consideration of potential contamination or large scale insertions with respect to the refer- ence. A record may align uniquely and have no close second match, but having a high number of mismatches may still imply that the reference is not the correct site. However we do not recommend use of this parameter unless you fully understand the impact of it and have determined that it is appro- priate for your sequencing technology. -D, --full-BAQ Run the BAQ algorithm on all reads, not just those in problematic re- gions. This matches the behaviour for Bcftools 1.12 and earlier. By default mpileup uses heuristics to decide when to apply the BAQ al- gorithm. Most sequences will not be BAQ adjusted, giving a CPU time closer to --no-BAQ, but it will still be applied in regions with sus- pected problematic alignments. This has been tested to work well on single sample data with even allele frequency, but the reliability is unknown for multi-sample calling and for low allele frequency variants so full BAQ is still recommended in those scenarios. -d, --max-depth INT At a position, read maximally INT reads per input file. Note that the original samtools mpileup command had a minimum value of 8000/n where n was the number of input files given to mpileup. This means that in sam- tools mpileup the default was highly likely to be increased and the -d parameter would have an effect only once above the cross-sample minimum of 8000. This behavior was problematic when working with a combination of single- and multi-sample bams, therefore in bcftools mpileup the user is given the full control (and responsibility), and an informative message is printed instead. Passing zero for this option sets it to the highest possible value, effectively removing the depth limit [250] -E, --redo-BAQ Recalculate BAQ on the fly, ignore existing BQ tags -f, --fasta-ref FILE The faidx-indexed reference file in the FASTA format. The file can be optionally compressed by bgzip. Reference is required by default unless the --no-reference option is set [null] --no-reference Do not require the --fasta-ref option. -G, --read-groups FILE list of read groups to include or exclude if prefixed with "^". One read group per line. This file can also be used to assign new sample names to read groups by giving the new sample name as a second white-space-separated field, like this: "read_group_id new_sam- ple_name". If the read group name is not unique, also the bam file name can be included: "read_group_id file_name sample_name". If all reads from the alignment file should be treated as a single sample, the as- terisk symbol can be used: "* file_name sample_name". Alignments with- out a read group ID can be matched with "?". NOTE: The meaning of bcftools mpileup -G is the opposite of samtools mpileup -G. RG_ID_1 RG_ID_2 SAMPLE_A RG_ID_3 SAMPLE_A RG_ID_4 SAMPLE_B RG_ID_5 FILE_1.bam SAMPLE_A RG_ID_6 FILE_2.bam SAMPLE_A * FILE_3.bam SAMPLE_C ? FILE_3.bam SAMPLE_D --indels-2.0 A new EXPERIMENTAL indel calling model which aims to address some known deficiencies of the current indel calling algorithm. Specifically, it uses diploid reference consensus sequence. Note that in the current version it has the potential to increase sensitivity but at the cost of decreased specificity. Only works with short-read sequencing technolo- gies. --indels-cns Another EXPERIMENTAL indel calling method, predating indels-2.0 in PR form, but merged more recently. It also uses a diploid reference con- sensus, but with added parameters and heuristics to optimise for a va- riety of sequencing platforms. This is usually faster and more accurate than the default caller and --indels-2.0, but has not been tested on non-diploid samples and samples without approximately even allele fre- quency. --no-indels-cns May be used to turn off --indels-cns mode when using one of the newer profiles that has this enabled by default. -q, -min-MQ INT Minimum mapping quality for an alignment to be used [0] -Q, --min-BQ INT Minimum base quality for a base to be considered [13] --max-BQ INT Caps the base quality to a maximum value [60]. This can be particularly useful on technologies that produce overly optimistic high qualities, leading to too many false positives or incorrect genotype assignments. -r, --regions CHR|CHR:POS|CHR:FROM-TO|CHR:FROM-[,...] Only generate mpileup output in given regions. Requires the alignment files to be indexed. If used in conjunction with -l then considers the intersection; see Common Options -R, --regions-file FILE As for -r, --regions, but regions read from FILE; see Common Options --regions-overlap 0|1|2 see Common Options --ignore-RG Ignore RG tags. Treat all reads in one alignment file as one sample. --ls, --skip-all-set Skip reads with all of the FLAG bits set [null] --ns, --skip-any-set Skip reads with any of the FLAG bits set. This option replaces and is synonymous to the deprecated --ff, --excl-flags [UNMAP,SECONDARY,QC- FAIL,DUP] --lu, --skip-all-unset Skip reads with all of the FLAG bits unset. This option replaces and is synonymous to the deprecated --rf, --incl-flags [null] --nu, --skip-any-unset Skip reads with any of the FLAG bits unset [null] -s, --samples LIST list of sample names. See Common Options -S, --samples-file FILE file of sample names to include or exclude if prefixed with "^". One sample per line. This file can also be used to rename samples by giving the new sample name as a second white-space-separated column, like this: "old_name new_name". If a sample name contains spaces, the spaces can be escaped using the backslash character, for example "Not\ a\ good\ sample\ name". -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options -x, --ignore-overlaps Disable read-pair overlap detection. --seed INT Set the random number seed used when sub-sampling deep regions [0]. Output options -a, --annotate LIST Comma-separated list of FORMAT and INFO tags to output. (case-insensi- tive, the "FORMAT/" prefix is optional, and use "?" to list available annotations on the command line) [null]: FORMAT/AD .. Allelic depth (Number=R,Type=Integer) FORMAT/ADF .. Allelic depths on the forward strand (Number=R,Type=Integer) FORMAT/ADR .. Allelic depths on the reverse strand (Number=R,Type=Integer) FORMAT/DP .. Number of high-quality bases (Number=1,Type=Integer) FORMAT/SP .. Phred-scaled strand bias P-value (Number=1,Type=Integer) FORMAT/SCR .. Number of soft-clipped reads (Number=1,Type=Integer) INFO/AD .. Total allelic depth (Number=R,Type=Integer) INFO/ADF .. Total allelic depths on the forward strand (Number=R,Type=Integer) INFO/ADR .. Total allelic depths on the reverse strand (Number=R,Type=Integer) INFO/SCR .. Number of soft-clipped reads (Number=1,Type=Integer) FORMAT/DV .. Deprecated in favor of FORMAT/AD; Number of high-quality non-reference bases, (Number=1,Type=Integer) FORMAT/DP4 .. Deprecated in favor of FORMAT/ADF and FORMAT/ADR; Number of high-quality ref-forward, ref-reverse, alt-forward and alt-reverse bases (Number=4,Type=Integer) FORMAT/DPR .. Deprecated in favor of FORMAT/AD; Number of high-quality bases for each observed allele (Number=R,Type=Integer) INFO/DPR .. Deprecated in favor of INFO/AD; Number of high-quality bases for each observed allele (Number=R,Type=Integer) -g, --gvcf INT[,...] output gVCF blocks of homozygous REF calls, with depth (DP) ranges specified by the list of integers. For example, passing 5,15 will group sites into two types of gVCF blocks, the first with minimum per-sample DP from the interval [5,15) and the latter with minimum depth 15 or more. In this example, sites with minimum per-sample depth less than 5 will be printed as separate records, outside of gVCF blocks. --no-version see Common Options -o, --output FILE Write output to FILE, rather than the default of standard output. (The same short option is used for both --open-prob and --output. If -o's argument contains any non-digit characters other than a leading + or - sign, it is interpreted as --output. Usually the filename extension will take care of this, but to write to an entirely numeric filename use -o ./123 or --output 123.) -O, --output-type b|u|z|v[0-9] see Common Options --threads INT see Common Options -U, --mwu-u The the previous Mann-Whitney U test score from version 1.12 and ear- lier. This is a probability score, but importantly it folds probabili- ties above or below the desired score into the same P. The new Mann-Whitney U test score is a "Z score", expressing the score as the number of standard deviations away from the mean (with zero being matching the mean). It keeps both positive and negative values. This can be important for some tests where errors are asymmetric. This option changes the INFO field names produced back to the ones used by the earlier Bcftools releases. For excample BQBZ becomes BQB. -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Options for SNP/INDEL genotype likelihood computation -X, --config STR Specify a platform specific configuration profile. Specifying the pro- file as "list" will list the available profile names and the parameters they change. There are profiles named after a release, which should be used if you wish to ensure forward compatibility of results. The non-versioned names (eg "illumina") will always point to the most re- cent set of parameters for that instrument type. The current values are: 1.12 -Q13 -h100 -m1 bgi bgi-1.20 --indels-cns -B --indel-size 80 -F0.1 --indel-bias 0.9 --seqq-offset 120 illumina-1.18 [ default values ] illumina illumina-1.20 --indels-cns --seqq-offset 125 ont -B -Q5 --max-BQ 30 -I ont-sup ont-sup-1.20 --indels-cns -B -Q1 --max-BQ 35 --delta-BQ 99 -F0.2 -o15 -e1 -h110 --del-bias 0.4 --indel-bias 0.7 --poly-mqual --seqq-offset 130 --indel-size 80 pacbio-ccs-1.18 -D -Q5 --max-BQ 50 -F0.1 -o25 -e1 -M99999 pacbio-ccs pacbio-ccs-1.20 --indels-cns -B -Q5 --max-BQ 50 -F0.1 -o25 -e1 -h300 --delta-BQ 10 --del-bias 0.4 --poly-mqual --indel-bias 0.9 --seqq-offset 118 --indel-size 80 --score-vs-ref 0.7 ultima ultima-1.20 --indels-cns -B -Q1 --max-BQ 30 --delta-BQ 10 -F0.15 -o20 -e10 -h250 --del-bias 0.3 --indel-bias 0.7 --poly-mqual --seqq-offset 140 --score-vs-ref 0.3 --indel-size 80 --ar, --ambig-reads drop|incAD|incAD0 What to do with ambiguous indel reads that do not span an entire short tandem repeat region: discard ambiguous reads from calling and do not increment high-quality AD depth counters (drop), exclude from calling but increment AD counters proportionally (incAD), exclude from calling and increment the first value of the AD counter (incAD0) [drop] -e, --ext-prob INT Phred-scaled gap extension sequencing error probability. Reducing INT leads to longer indels [20] -F, --gap-frac FLOAT Minimum fraction of gapped reads [0.002] -h, --tandem-qual INT Coefficient for modeling homopolymer errors. Given an l-long homopoly- mer run, the sequencing error of an indel of size s is modeled as INT*s/l [500] Increasing this informs the caller that indels in long homopolymers are more likely genuine and less likely to be sequencing artifacts. Hence increasing tandem-qual will have higher recall and lower precision. Bcftools 1.12 and earlier had a default of 100, which was tuned around more error prone instruments. Note changing this may have a minor impact on SNP calling too. For maximum SNP calling accu- racy, it may be preferable to adjust this lower again, although this will adversely affect indels. --indel-bias FLOAT Skews the indel scores up or down, trading recall (low false-negative) vs precision (low false-positive) [1.0]. In Bcftools 1.12 and earlier this parameter didn't exist, but had an implied value of 1.0. If you are planning to do heavy filtering of variants, selecting the best quality ones only (favouring precision over recall), it is advisable to set this lower (such as 0.75) while higher depth samples or where you favour recall rates over precision may work better with a higher value such as 2.0. --del-bias FLOAT Skews the likelihood of deletions over insertions. Defaults to an even distribution value of 1.0. Lower values imply a higher rate of false positive deletions (meaning candidate deletions are less likely to be real). --indel-size INT Indel window size to use when assessing the quality of candidate in- dels. Note that although the window size approximately corresponds to the maximum indel size considered, it is not an exact threshold [110] --seqq-offset INT Tunes the importance of indel sequence quality per depth. The final "seqQ" quality used is "offset - 5*MIN(depth,20)". [120] --poly-mqual Use the lowest quality value within a homopolymer run, instead of the quality immediately adjacent to the indel. This may be important for unclocked instruments, particularly ones with a flow chemistry where runs of bases of identical type are incorporated together. -I, --skip-indels Do not perform INDEL calling -L, --max-idepth INT Skip INDEL calling if the average per-sample depth is above INT [250] -m, --min-ireads INT Minimum number gapped reads for indel candidates INT [1] -M, --max-read-len INT The maximum read length permitted by the BAQ algorithm [500]. Variants are still called on longer reads, but they will not be passed through the BAQ method. This limit exists to prevent excessively long BAQ times and high memory usage. Note if partial BAQ is enabled with -D then raising this parameter will likely not have a significant a CPU cost. -o, --open-prob INT Phred-scaled gap open sequencing error probability. Reducing INT leads to more indel calls. (The same short option is used for both --open-prob and --output. When -o's argument contains only an optional + or - sign followed by the digits 0 to 9, it is interpreted as --open-prob.) [40] -p, --per-sample-mF Apply -m and -F thresholds per sample to increase sensitivity of call- ing. By default both options are applied to reads pooled from all sam- ples. -P, --platforms STR Comma-delimited list of platforms (determined by @RG-PL) from which indel candidates are obtained. It is recommended to collect indel can- didates from sequencing technologies that have low indel error rate such as ILLUMINA [all] Examples: Call SNPs and short INDELs, then mark low quality sites and sites with the read depth exceeding a limit. (The read depth should be adjusted to about twice the average read depth as higher read depths usually indicate prob- lematic regions which are often enriched for artefacts.) One may consider to add -C50 to mpileup if mapping quality is overestimated for reads con- taining excessive mismatches. Applying this option usually helps for BWA-backtrack alignments, but may not other aligners. bcftools mpileup -Ou -f ref.fa aln.bam | \ bcftools call -Ou -mv | \ bcftools filter -s LowQual -e 'QUAL<20 || DP>100' > var.flt.vcf bcftools norm [OPTIONS] file.vcf.gz Left-align and normalize indels, check if REF alleles match the reference, split multiallelic sites into multiple rows; recover multiallelics from multiple rows. Left-alignment and normalization will only be applied if the --fasta-ref option is supplied. -a, --atomize Decompose complex variants, e.g. split MNVs into consecutive SNVs. See also --atom-overlaps and --old-rec-tag. --atom-overlaps .|* Alleles missing because of an overlapping variant can be set either to missing (.) or to the star alele (*), as recommended by the VCF speci- fication. IMPORTANT: Note that asterisk is expaneded by shell and must be put in quotes or escaped by a backslash: # Before atomization: 100 CC C,GG 1/2 # After: # bcftools norm -a --atom-overlaps . 100 C G ./1 100 CC C 1/. 101 C G ./1 # After: # bcftools norm -a --atom-overlaps '*' # bcftools norm -a --atom-overlaps \* 100 C G,* 2/1 100 CC C,* 1/2 101 C G,* 2/1 -c, --check-ref e|w|x|s what to do when incorrect or missing REF allele is encountered: exit (e), warn (w), exclude (x), or set/fix (s) bad sites. The w option can be combined with x and s. Note that s can swap alleles and will update genotypes (GT) and AC counts, but will not attempt to fix PL or other fields. Also note, and this cannot be stressed enough, that s will NOT fix strand issues in your VCF, do NOT use it for that purpose!!! (In- stead see http://samtools.github.io/bcftools/howtos/plugin.af-dist.html and http://samtools.github.io/bcftools/howtos/plugin.fixref.html.) -d, --rm-dup snps|indels|both|all|exact If a record is present multiple times, output only the first instance. See also --collapse in Common Options. -D, --remove-duplicates If a record is present in multiple files, output only the first in- stance. Alias for -d exact, deprecated. -e, --exclude EXPRESSION do not normalize input records for which EXPRESSION is true. For valid expressions see EXPRESSIONS. Note that duplicate removal ignores this option. -f, --fasta-ref FILE reference sequence. Supplying this option will turn on left-alignment and normalization, however, see also the --do-not-normalize option be- low. --force try to proceed with -m- even if malformed tags with incorrect number of fields are encountered, discarding such tags. (Experimental, use at your own risk.) -g, --gff-annot FILE when a GFF file is provided, follow HGVS 3'rule and right-align vari- ants in transcripts on the forward strand. In case of overlapping tran- scripts, the default mode is to left-align the variant. For a descrip- tion of the supported GFF3 file format see bcftools csq. -i, --include EXPRESSION normalize only input records for which EXPRESSION is true. For valid expressions see EXPRESSIONS. Note that duplicate removal ignores this option. --keep-sum TAG[,...] keep vector sum constant when splitting multiallelic sites. Only AD tag is currently supported. See also https://github.com/samtools/bcftools/issues/360 -m, --multiallelics -|+[snps|indels|both|any] split multiallelic sites into biallelic records (-) or join biallelic sites into multiallelic records (+). An optional type string can follow which controls variant types which should be split or merged together: If only SNP records should be split or merged, specify snps; if both SNPs and indels should be merged separately into two records, specify both; if SNPs and indels should be merged into a single record, specify any. Note that multiallelic sites with both SNPs and indels will be split into biallelic sites with both -m -snps and -m -indels. --multi-overlaps 0|. use the reference (0) or missing (.) allele for overlapping alleles af- ter splitting multiallelic sites --no-version see Common Options -N, --do-not-normalize the -c s option can be used to fix or set the REF allele from the ref- erence -f. The -N option will not turn on indel normalisation as the -f option normally implies --old-rec-tag STR Add INFO/STR annotation with the original record. The format of the an- notation is CHROM|POS|REF|ALT|USED_ALT_IDX. -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --strict-filter when merging (-m+), merged site is PASS only if all sites being merged PASS -S, --sort pos|lex when splitting sites or processing duplicates, sort records on output by POS only (pos, the default) or by POS and lexicographically by REF+ALT (lex) -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options --threads INT see Common Options -v, --verbosity INT see Common Options -w, --site-win INT maximum distance between two records to consider when locally sorting variants which changed position during the realignment -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. bcftools [plugin NAME|+NAME] [OPTIONS] FILE -- [PLUGIN OPTIONS] A common framework for various utilities. The plugins can be used the same way as normal commands only their name is prefixed with "+". Most plugins accept two types of parameters: general options shared by all plugins fol- lowed by a separator, and a list of plugin-specific options. There are some exceptions to this rule, some plugins do not accept the common options and implement their own parameters. Therefore please pay attention to the usage examples that each plugin comes with. VCF input options: -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options VCF output options: --no-version see Common Options -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options --threads INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Plugin options: -h, --help list plugin's options -l, --list-plugins List all available plugins. By default, appropriate system directories are searched for installed plugins. You can override this by setting the BCFTOOLS_PLUGINS environ- ment variable to a colon-separated list of directories to search. If BCFTOOLS_PLUGINS begins with a colon, ends with a colon, or contains adjacent colons, the system directories are also searched at that posi- tion in the list of directories. -v, --verbosity INT see Common Options -V, --version print version string and exit List of plugins coming with the distribution: ad-bias find positions with wildly varying ALT allele frequency (Fisher test on FMT/AD) add-variantkey add VariantKey INFO fields VKX and RSX af-dist collect AF deviation stats and GT probability distribution given AF and assuming HWE vrfs assess site noisiness (variant read frequency score) from a large num- ber of unaffected parental samples allele-length count the frequency of the length of REF, ALT and REF+ALT check-ploidy check if ploidy of samples is consistent for all sites check-sparsity print samples without genotypes in a region or chromosome color-chrs color shared chromosomal segments, requires trio VCF with phased GTs contrast runs a basic association test, per-site or in a region, and checks for novel alleles and genotypes in two groups of samples. Adds the follow- ing INFO annotations: * PASSOC .. Fisher's exact test probability of genotypic association (REF vs non-REF allele) * FASSOC .. proportion of non-REF allele in controls and cases * NASSOC .. number of control-ref, control-alt, case-ref and case-alt alleles * NOVELAL .. lists samples with a novel allele not observed in the control group * NOVELGT .. lists samples with a novel genotype not observed in the control group counts a minimal plugin which counts number of SNPs, Indels, and total number of sites. dosage print genotype dosage. By default the plugin searches for PL, GL and GT, in that order. fill-from-fasta fill INFO or REF field based on values in a fasta file fill-tags set various INFO tags. The list of tags supported in this version: * INFO/AC Number:A Type:Integer .. Allele count in geno- types * INFO/AC_Hom Number:A Type:Integer .. Allele counts in homozy- gous genotypes * INFO/AC_Het Number:A Type:Integer .. Allele counts in het- erozygous genotypes * INFO/AC_Hemi Number:A Type:Integer .. Allele counts in hemizy- gous genotypes * INFO/AF Number:A Type:Float .. Allele frequency * INFO/AN Number:1 Type:Integer .. Total number of alleles in called genotypes * INFO/ExcHet Number:A Type:Float .. Test excess heterozygos- ity; 1=good, 0=bad * INFO/END Number:1 Type:Integer .. End position of the variant * INFO/F_MISSING Number:1 Type:Float .. Fraction of missing genotypes * INFO/HWE Number:A Type:Float .. HWE test (PMID:15789306); 1=good, 0=bad * INFO/MAF Number:A Type:Float .. Minor Allele frequency * INFO/NS Number:1 Type:Integer .. Number of samples with data * INFO/TYPE Number:. Type:String .. The record type (REF,SNP,MNP,INDEL,etc) * FORMAT/VAF Number:A Type:Float .. The fraction of reads with the alternate allele, requires FORMAT/AD or ADF+ADR * FORMAT/VAF1 Number:1 Type:Float .. The same as FORMAT/VAF but for all alternate alleles cumulatively * TAG=func(TAG) Number:1 Type:Integer .. Experimental support for user-defined expressions such as "DP=sum(DP)" fixploidy sets correct ploidy fixref determine and fix strand orientation frameshifts annotate frameshift indels GTisec count genotype intersections across all possible sample subsets in a vcf file GTsubset output only sites where the requested samples all exclusively share a genotype guess-ploidy determine sample sex by checking genotype likelihoods (GL,PL) or geno- types (GT) in the non-PAR region of chrX. gvcfz compress gVCF file by resizing non-variant blocks according to speci- fied criteria impute-info add imputation information metrics to the INFO field based on selected FORMAT tags indel-stats calculates per-sample or de novo indels stats. The usage and format is similar to smpl-stats and trio-stats isecGT compare two files and set non-identical genotypes to missing mendelian count Mendelian consistent / inconsistent genotypes (deprecated, use +mendelian2 instead) mendelian2 count Mendelian consistent / inconsistent genotypes. missing2ref sets missing genotypes ("./.") to ref allele ("0/0" or "0|0") parental-origin determine parental origin of a CNV region prune annotate sites with or prune sites by the number of variants within a window, Lewontin's D (doi:10.1093/molbev/msz265), Ragsdale's D (doi:10.1534/genetics.108.093153), or correlation coefficient r-squared. remove-overlaps remove overlapping variants and duplicate sites scatter intended as an inverse to bcftools concat, scatter VCF by chunks or re- gions, creating multiple VCFs. setGT general tool to set genotypes according to rules requested by the user smpl-stats calculates basic per-sample stats. The usage and format is similar to indel-stats and trio-stats. split split VCF by sample, creating single- or multi-sample VCFs split-vep extract fields from structured annotations such as INFO/CSQ created by VEP or INFO/BCSQ created by bcftools/csq. These can be added as a new INFO field to the VCF or in a custom text format. See http://samtools.github.io/bcftools/howtos/plugin.split-vep.html for more. tag2tag Convert between similar tags, such as GL,PL,GP or QR,QA,QS or tags with localized alleles e.g. LPL,LAD. See http://samtools.github.io/bcftools/howtos/plugin.tag2tag.html for more. trio-dnm2 screen variants for possible de-novo mutations in trios trio-stats calculate transmission rate in trio children. The usage and format is similar to indel-stats and smpl-stats. trio-switch-rate calculate phase switch rate in trio samples, children samples must have phased GTs variant-distance annotate sites with the distance to the nearest variant variantkey-hex generate unsorted VariantKey-RSid index files in hexadecimal format vcf2table print the variants as a set of tables Examples: # List options common to all plugins bcftools plugin # List available plugins bcftools plugin -l # Run a plugin bcftools plugin counts in.vcf # Run a plugin using the abbreviated "+" notation bcftools +counts in.vcf # Run a plugin from an explicit location bcftools +/path/to/counts.so in.vcf # The input VCF can be streamed just like in other commands cat in.vcf | bcftools +counts # Print usage information of plugin "dosage" bcftools +dosage -h # Replace missing genotypes with 0/0 bcftools +missing2ref in.vcf # Replace missing genotypes with 0|0 bcftools +missing2ref in.vcf -- -p Plugins troubleshooting: Things to check if your plugin does not show up in the bcftools plugin -l output: * Run with the -v option for verbose output: bcftools plugin -lv * Does the environment variable BCFTOOLS_PLUGINS include the correct path? Plugins API: // Short description used by 'bcftools plugin -l' const char *about(void); // Longer description used by 'bcftools +name -h' const char *usage(void); // Called once at startup, allows initialization of local variables. // Return 1 to suppress normal VCF/BCF header output, -1 on critical // errors, 0 otherwise. int init(int argc, char **argv, bcf_hdr_t *in_hdr, bcf_hdr_t *out_hdr); // Called for each VCF record, return NULL to suppress the output bcf1_t *process(bcf1_t *rec); // Called after all lines have been processed to clean up void destroy(void); bcftools polysomy [OPTIONS] file.vcf.gz Detect number of chromosomal copies in VCFs annotates with the Illumina's B-allele frequency (BAF) values. Note that this command is not compiled in by default, see the section Optional Compilation with GSL in the INSTALL file for help. General options: -o, --output-dir path output directory -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --sample string sample name -t, --targets LIST see Common Options -T, --targets-file FILE see Common Options --targets-overlap 0|1|2 see Common Options -v, --verbosity INT verbose debugging output which gives hints about the thresholds and de- cisions made by the program. Note that the exact output can change be- tween versions. See also Common Options Algorithm options: -b, --peak-size float the minimum peak size considered as a good match can be from the inter- val [0,1] where larger is stricter -c, --cn-penalty float a penalty for increasing copy number state. How this works: multiple peaks are always a better fit than a single peak, therefore the program prefers a single peak (normal copy number) unless the absolute devia- tion of the multiple peaks fit is significantly smaller. Here the mean- ing of "significant" is given by the float from the interval [0,1] where larger is stricter. -f, --fit-th float threshold for goodness of fit (normalized absolute deviation), smaller is stricter -i, --include-aa include also the AA peak in CN2 and CN3 evaluation. This usually re- quires increasing -f. -m, --min-fraction float minimum distinguishable fraction of aberrant cells. The experience shows that trustworthy are estimates of 20% and more. -p, --peak-symmetry float a heuristics to filter failed fits where the expected peak symmetry is violated. The float is from the interval [0,1] and larger is stricter bcftools query [OPTIONS] file.vcf.gz [file.vcf.gz [...]] Extracts fields from VCF or BCF files and outputs them in user-defined for- mat. -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. --force-samples continue even when some samples requested via -s/-S do not exist -f, --format FORMAT learn by example, see below -F, --print-filtered STR by default, samples failing -i/-e filtering expressions are suppressed from output when FORMAT fields are queried (for example %CHROM %POS [ %GT]). With -F, such fields will be still printed but instead of their actual value, STR will be used. -H, --print-header print header. By default, the header is printed with column indices, e.g. "#[1]CHROM". These can be suppressed by giving the option twice, "-HH". -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -l, --list-samples list sample names and exit -N, --disable-automatic-newline disable automatic addition of a missing newline character at the end of the formatting expression. By default, the program checks if the ex- pression contains a newline and appends it if not, to prevent format- ting the entire output into a single line by mistake. Note that ver- sions prior to 1.18 had no automatic check and newline had to be in- cluded explicitly. -o, --output FILE see Common Options -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST see Common Options -S, --samples-file FILE see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options -u, --allow-undef-tags do not throw an error if there are undefined tags in the format string, print "." instead -v, --vcf-list FILE process multiple VCFs listed in the file --verbosity INT see Common Options Format: %CHROM The CHROM column (similarly also other columns: POS, ID, REF, ALT, QUAL, FILTER) %END End position of the REF allele %END0 End position of the REF allele in 0-based coordinates %FIRST_ALT Alias for %ALT{0} %FORMAT Prints all FORMAT fields or a subset of samples with -s or -S %GT Genotype (e.g. 0/1) %FUNCTION Functions supported by the -i/-e filtering expressions (e.g. "[ %sSUM(FMT/AD)] %SUM(FMT/AD) %SUM(INFO/AD)") %INFO Prints the whole INFO column %INFO/TAG Any tag in the INFO column %IUPACGT Genotype translated to IUPAC ambiguity codes (e.g. M instead of C/A) %LINE Prints the whole line %MASK Indicates presence of the site in other files (with multiple files) %N_PASS(expr) Number of samples that pass the filtering expression (see *<<expressions,EXPRESSIONS>>*) %POS0 POS in 0-based coordinates %PBINOM(TAG) Calculate phred-scaled binomial probability, the allele index is determined from GT %SAMPLE Sample name %TAG{INT} Curly brackets to print a subfield (e.g. INFO/TAG{1}, the indexes are 0-based) %TBCSQ Translated FORMAT/BCSQ. See the csq command above for explanation and examples. %TGT Translated genotype (e.g. C/A) %TYPE Variant type (REF, SNP, MNP, INDEL, BND, OTHER) %VKX VariantKey, biallelic hexadecimal encoding of CHROM,POS,REF,ALT (https://github.com/tecnickcom/variantkey) [] Format fields must be enclosed in brackets to loop over all samples \n new line \t tab character Everything else is printed verbatim. Examples: # Print chromosome, position, ref allele and the first alternate allele bcftools query -f '%CHROM %POS %REF %ALT{0}\n' file.vcf.gz # Similar to above, but use tabs instead of spaces, add sample name and genotype bcftools query -f '%CHROM\t%POS\t%REF\t%ALT[\t%SAMPLE=%GT]\n' file.vcf.gz # Print FORMAT/GT fields followed by FORMAT/GT fields bcftools query -f 'GQ:[ %GQ] \t GT:[ %GT]\n' file.vcf # Make a BED file: chr, pos (0-based), end pos (1-based), id bcftools query -f'%CHROM\t%POS0\t%END\t%ID\n' file.bcf # Print only samples with alternate (non-reference) genotypes bcftools query -f'[%CHROM:%POS %SAMPLE %GT\n]' -i'GT="alt"' file.bcf # Print all samples at sites with at least one alternate genotype bcftools view -i'GT="alt"' file.bcf -Ou | bcftools query -f'[%CHROM:%POS %SAMPLE %GT\n]' # Print phred-scaled binomial probability from FORMAT/AD tag for all heterozygous genotypes bcftools query -i'GT="het"' -f'[%CHROM:%POS %SAMPLE %GT %PBINOM(AD)\n]' file.vcf # Print the second value of AC field if bigger than 10. Note the (unfortunate) difference in # index subscript notation: formatting expressions (-f) uses "{}" while filtering expressions # (-i) use "[]". This is for historic reasons and backward-compatibility. bcftools query -f '%AC{1}\n' -i 'AC[1]>10' file.vcf.gz # Print all samples at sites where at least one sample has DP=1 or DP=2. In the second case # print only samples with DP=1 or DP=2, the difference is in the logical operator used, || vs |. bcftools query -f '[%SAMPLE %GT %DP\n]' -i 'FMT/DP=1 || FMT/DP=2' file.vcf bcftools query -f '[%SAMPLE %GT %DP\n]' -i 'FMT/DP=1 | FMT/DP=2' file.vcf # Refer to ID column vs INFO/ID tag vs FORMAT/ID tag bcftools query -f 'columnID=%ID infoID=%INFO/ID [fmtID=%ID ] [columnID=%/ID]' bcftools reheader [OPTIONS] file.vcf.gz Modify header of VCF/BCF files, change sample names. -f, --fai FILE add to the header contig names and their lengths from the provided fasta index file (.fai). Lengths of existing contig lines will be up- dated and contig lines not present in the fai file will be removed -h, --header FILE new VCF header -n, --samples-list LIST comma-separated list of new sample names -s, --samples FILE, -N, --samples-file FILE new sample names, one name per line, in the same order as they appear in the VCF file. Alternatively, only samples which need to be renamed can be listed as "old_name new_name\n" pairs separated by whitespaces, each on a separate line. If a sample name contains spaces, the spaces can be escaped using the backslash character, for example "Not\ a\ good\ sample\ name". -o, --output FILE see Common Options -T, --temp-prefix PATH this option is ignored, but left for compatibility with earlier ver- sions of bcftools. --threads INT see Common Options -v, --verbosity INT see Common Options bcftools roh [OPTIONS] file.vcf.gz A program for detecting runs of homo/autozygosity. Only bi-allelic sites are considered. The results can be visualized in HTML/JavaScript using the roh-viz script. The HMM model: Notation: D = Data, AZ = autozygosity, HW = Hardy-Weinberg (non-autozygosity), f = non-ref allele frequency Emission probabilities: oAZ = P_i(D|AZ) = (1-f)*P(D|RR) + f*P(D|AA) oHW = P_i(D|HW) = (1-f)^2 * P(D|RR) + f^2 * P(D|AA) + 2*f*(1-f)*P(D|RA) Transition probabilities: tAZ = P(AZ|HW) .. from HW to AZ, the -a parameter tHW = P(HW|AZ) .. from AZ to HW, the -H parameter ci = P_i(C) .. probability of cross-over at site i, from genetic map AZi = P_i(AZ) .. probability of site i being AZ/non-AZ, scaled so that AZi+HWi = 1 HWi = P_i(HW) P_{i+1}(AZ) = oAZ * max[(1 - tAZ * ci) * AZ{i-1} , tAZ * ci * (1-AZ{i-1})] P_{i+1}(HW) = oHW * max[(1 - tHW * ci) * (1-AZ{i-1}) , tHW * ci * AZ{i-1}] General Options: --AF-dflt FLOAT in case allele frequency is not known, use the FLOAT. By default, sites where allele frequency cannot be determined, or is 0, are skipped. --AF-tag TAG use the specified INFO tag TAG as an allele frequency estimate instead of the default AC and AN tags. Sites which do not have TAG will be skipped. --AF-file FILE Read allele frequencies from a tab-delimited file containing the columns: CHROM\tPOS\tREF,ALT\tAF. The file can be compressed with bgzip and indexed with tabix -s1 -b2 -e2. Sites which are not present in the FILE or have different reference or alternate allele will be skipped. Note that such a file can be easily created from a VCF using: bcftools query -f'%CHROM\t%POS\t%REF,%ALT\t%INFO/TAG\n' file.vcf | bgzip -c > freqs.tab.gz -b, --buffer-size INT[,INT] when the entire many-sample file cannot fit into memory, a sliding buffer approach can be used. The first value is the number of sites to keep in memory. If negative, it is interpreted as the maximum memory to use, in MB. The second, optional, value sets the number of overlapping sites. The default overlap is set to roughly 1% of the buffer size. -e, --estimate-AF FILE estimate the allele frequency by recalculating INFO/AC and INFO/AN on the fly, using the specified TAG which can be either FORMAT/GT ("GT") or FORMAT/PL ("PL"). If TAG is not given, "GT" is assumed. Either all samples ("-") or samples listed in FILE will be included. For example, use "PL,-" to estimate AF from FORMAT/PL of all samples. If neither -e nor the other --AF-... options are given, the allele frequency is esti- mated from AC and AN counts which are already present in the INFO field. --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -G, --GTs-only FLOAT use genotypes (FORMAT/GT fields) ignoring genotype likelihoods (FOR- MAT/PL), setting PL of unseen genotypes to FLOAT. Safe value to use is 30 to account for GT errors. --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -I, --skip-indels skip indels as their genotypes are usually enriched for errors -m, --genetic-map FILE genetic map in the format required also by IMPUTE2. Only the first and third column are used (position and Genetic_Map(cM)). The FILE can be a single file or a file mask, where string "{CHROM}" is replaced with chromosome name. -M, --rec-rate FLOAT constant recombination rate per bp. In combination with --genetic-map, the --rec-rate parameter is interpreted differently, as FLOAT-fold in- crease of transition probabilities, which allows the model to become more sensitive yet still account for recombination hotspots. Note that also the range of the values is therefore different in both cases: nor- mally the parameter will be in the range (1e-3,1e-9) but with --ge- netic-map it will be in the range (10,1000). -o, --output FILE Write output to the FILE, by default the output is printed on stdout -O, --output-type s|r[z] Generate per-site output (s) or per-region output (r). By default both types are printed and the output is uncompressed. Add z for a com- pressed output. # Output fields: RG = predicted homo/autozygous regions - Sample - Chromosome - Start - End - Length (bp) - Number of markers - Quality .. average phred score in the region from the forward-backward algorithm ST = per-site output showing: - Sample - Chromosome - Position - State .. predicted state from the Viterbi algorithm, 0 for normal (HW, Hardy-Weinberg) or 1 for autozygous (AZ) - Quality .. quality score from the forward-backward algorithm -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST see Common Options -S, --samples-file FILE see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options -v, --verbosity INT see Common Options HMM Options: -a, --hw-to-az FLOAT P(AZ|HW) transition probability from AZ (autozygous) to HW (Hardy-Wein- berg) state -H, --az-to-hw FLOAT P(HW|AZ) transition probability from HW to AZ state -V, --viterbi-training FLOAT estimate HMM parameters using Baum-Welch algorithm, using the conver- gence threshold FLOAT, e.g. 1e-10 (experimental) bcftools sort [OPTIONS] file.bcf -m, --max-mem FLOAT[kMG] Maximum memory to use. Approximate, affects the number of temporary files written to the disk. Note that if the command fails at this step because of too many open files, your system limit on the number of open files ("ulimit") may need to be increased. -o, --output FILE see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -T, --temp-dir DIR Use this directory to store temporary files. If the last six characters of the string DIR are XXXXXX, then these are replaced with a string that makes the directory name unique. -v, --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. bcftools stats [OPTIONS] A.vcf.gz [B.vcf.gz] Parses VCF or BCF and produces text file stats which is suitable for ma- chine processing and can be plotted using plot-vcfstats. When two files are given, the program generates separate stats for intersection and the com- plements. By default only sites are compared, -s/-S must given to include also sample columns. When one VCF file is specified on the command line, then stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc. are printed. When two VCF files are given, then stats such as concordance (Genotype concordance by non-reference allele frequency, Genotype concordance by sample, Non-Ref- erence Discordance) and correlation are also printed. Per-site discordance (PSD) is also printed in --verbose mode. --af-bins LIST|FILE comma separated list of allele frequency bins (e.g. 0.1,0.5,1) or a file listing the allele frequency bins one per line (e.g. 0.1\n0.5\n1) --af-tag TAG allele frequency INFO tag to use for binning. By default the allele frequency is estimated from AC/AN, if available, or directly from the genotypes (GT) if not. -1, --1st-allele-only consider only the 1st alternate allele at multiallelic sites -c, --collapse snps|indels|both|all|some|none see Common Options -d, --depth INT,INT,INT ranges of depth distribution: min, max, and size of the bin --debug produce verbose per-site and per-sample output -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -E, --exons file.gz tab-delimited file with exons for indel frameshifts statistics. The columns of the file are CHR, FROM, TO, with 1-based, inclusive, posi- tions. The file is BGZF-compressed and indexed with tabix tabix -s1 -b2 -e3 file.gz -f, --apply-filters LIST see Common Options -F, --fasta-ref ref.fa faidx indexed reference sequence file to determine INDEL context -i, --include EXPRESSION include only sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -I, --split-by-ID collect stats separately for sites which have the ID column set ("known sites") or which do not have the ID column set ("novel sites"). -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -s, --samples LIST see Common Options -S, --samples-file FILE see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options -u, --user-tstv <TAG[:min:max:n]> collect Ts/Tv stats for any tag using the given binning [0:1:100] -v, --verbosity INT see Common Options bcftools view [OPTIONS] file.vcf.gz [REGION [...]] View, subset and filter VCF or BCF files by position and filtering expres- sion. Convert between VCF and BCF. Former bcftools subset. Output options -G, --drop-genotypes drop individual genotype information (after subsetting if -s option is set) -h, --header-only output the VCF header only (see also bcftools head) -H, --no-header suppress the header in VCF output --with-header output both VCF header and records (this is the default, but the option is useful for explicitness or to reset the effects of -h or -H) -l, --compression-level [0-9] compression level. 0 stands for uncompressed, 1 for best speed and 9 for best compression. --no-version see Common Options -O, --output-type b|u|z|v[0-9] see Common Options -o, --output FILE: output file name. If not present, the default is to print to standard output (stdout). -r, --regions chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -R, --regions-file file see Common Options --regions-overlap 0|1|2 see Common Options -t, --targets chr|chr:pos|chr:from-to|chr:from-[,...] see Common Options -T, --targets-file file see Common Options --targets-overlap 0|1|2 see Common Options --threads INT see Common Options --verbosity INT see Common Options -W[FMT], -W[=FMT], --write-index[=FMT] Automatically index the output file. FMT is optional and can be one of "tbi" or "csi" depending on output file format. Subset options: -A, --trim-unseen-alleles remove the unseen allele <*> or <NON_REF> at variant sites when the op- tion is given once (-A) or at all sites when the options is given twice (-AA). -a, --trim-alt-alleles remove alleles not seen in the genotype fields from the ALT column. Note that if no alternate allele remains after trimming, the record it- self is not removed but ALT is set to ".". If the option -s or -S is given, removes alleles not seen in the subset. INFO and FORMAT tags de- clared as Type=A, G or R will be trimmed as well. --force-samples only warn about unknown subset samples -I, --no-update do not (re)calculate INFO fields for the subset (currently INFO/AC and INFO/AN) -s, --samples LIST see Common Options. Note that it is possible to create multiple subsets simultaneously using the split plugin. -S, --samples-file FILE see Common Options. Note that it is possible to create multiple subsets simultaneously using the split plugin. Filter options: Note that filter options below dealing with counting the number of alleles will, for speed, first check for the values of AC and AN in the INFO column to avoid parsing all the genotype (FORMAT/GT) fields in the VCF. This means that filters like --uncalled, --exclude-uncalled', or --min-af 0.1 will be calculated from INFO/AC and INFO/AN when available or FORMAT/GT otherwise. However, it will not attempt to use any other existing field, like INFO/AF for example. For that, use --exclude AF<0.1 instead. Also note that one must be careful when sample subsetting and filtering is performed in a single command because the order of internal operations can influence the result. For example, the -i/-e filtering is performed before sample removal, but the -P filtering is performed after, and some are in- herently ambiguous, for example allele counts can be taken from the INFO column when present but calculated on the fly when absent. Therefore it is strongly recommended to spell out the required order explicitly by separat- ing such commands into two steps. (Make sure to use the -O u option when piping!) -c, --min-ac INT[:nref|:alt1|:minor|:major|:'nonmajor'] minimum allele count (INFO/AC) of sites to be printed. Specifying the type of allele is optional and can be set to non-reference (nref, the default), 1st alternate (alt1), the least frequent (minor), the most frequent (major) or sum of all but the most frequent (nonmajor) alle- les. -C, --max-ac INT[:nref|:alt1|:minor|:'major'|:'nonmajor'] maximum allele count (INFO/AC) of sites to be printed. Specifying the type of allele is optional and can be set to non-reference (nref, the default), 1st alternate (alt1), the least frequent (minor), the most frequent (major) or sum of all but the most frequent (nonmajor) alle- les. -e, --exclude EXPRESSION exclude sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -f, --apply-filters LIST see Common Options -g, --genotype [^][hom|het|miss] include only sites with one or more homozygous (hom), heterozygous (het) or missing (miss) genotypes. When prefixed with ^, the logic is reversed; thus ^het excludes sites with heterozygous genotypes. -i, --include EXPRESSION include sites for which EXPRESSION is true. For valid expressions see EXPRESSIONS. -k, --known print known sites only (ID column is not ".") -m, --min-alleles INT print sites with at least INT alleles listed in REF and ALT columns -M, --max-alleles INT print sites with at most INT alleles listed in REF and ALT columns. Use -m2 -M2 -v snps to only view biallelic SNPs. -n, --novel print novel sites only (ID column is ".") -p, --phased print sites where all samples are phased. Haploid genotypes are consid- ered phased. Missing genotypes considered unphased unless the phased bit is set. -P, --exclude-phased exclude sites where all samples are phased -q, --min-af FLOAT[:nref|:alt1|:minor|:major|:nonmajor] minimum allele frequency (INFO/AC / INFO/AN) of sites to be printed. Specifying the type of allele is optional and can be set to non-refer- ence (nref, the default), 1st alternate (alt1), the least frequent (minor), the most frequent (major) or sum of all but the most frequent (nonmajor) alleles. -Q, --max-af FLOAT[:nref|:alt1|:minor|:major|:nonmajor] maximum allele frequency (INFO/AC / INFO/AN) of sites to be printed. Specifying the type of allele is optional and can be set to non-refer- ence (nref, the default), 1st alternate (alt1), the least frequent (minor), the most frequent (major) or sum of all but the most frequent (nonmajor) alleles. -u, --uncalled print sites without a called genotype, i.e. print sites with all geno- types missing. Note that the missingness is determined from INFO/AN and AC tags when available to avoid parsing sample fields. -U, --exclude-uncalled exclude sites without a called genotype, i.e. print sites with at least one non-missing genotype. Note that the missingness is determined from INFO/AN and AC tags when available to avoid parsing sample fields. -v, --types snps|indels|mnps|other comma-separated list of variant types to select. Site is selected if any of the ALT alleles is of the type requested. Types are determined by comparing the REF and ALT alleles in the VCF record not INFO tags like INFO/INDEL or INFO/VT. Use --include to select based on INFO tags. -V, --exclude-types snps|indels|mnps|ref|bnd|other comma-separated list of variant types to exclude. Site is excluded if any of the ALT alleles is of the type requested. Types are determined by comparing the REF and ALT alleles in the VCF record not INFO tags like INFO/INDEL or INFO/VT. Use --exclude to exclude based on INFO tags. -x, --private print sites where only the subset samples carry an non-reference al- lele. Requires --samples or --samples-file. -X, --exclude-private exclude sites where only the subset samples carry an non-reference al- lele bcftools help [COMMAND] | bcftools --help [COMMAND] Display a brief usage message listing the bcftools commands available. If the name of a command is also given, e.g., bcftools help view, the detailed usage message for that particular command is displayed. bcftools [--version|-v] Display the version numbers and copyright information for bcftools and the important libraries used by bcftools. bcftools [--version-only] Display the full bcftools version number in a machine-readable format. SCRIPTS gff2gff Attempts to fix a GFF file to be correctly parsed by csq. zcat in.gff.gz | gff2gff | gzip -c > out.gff.gz plot-vcfstats [OPTIONS] file.vchk [...] Script for processing output of bcftools stats. It can merge results from multiple outputs (useful when running the stats for each chromosome sepa- rately), plots graphs and creates a PDF presentation. -m, --merge Merge vcfstats files to STDOUT, skip plotting. -p, --prefix DIR The output directory. This directory will be created if it does not ex- ist. -P, --no-PDF Skip the PDF creation step. -r, --rasterize Rasterize PDF images for faster rendering. This is the default and the opposite of -v, --vectors. -s, --sample-names Use sample names for xticks rather than numeric IDs. -t, --title STRING Identify files by these titles in plots. The option can be given multi- ple times, for each ID in the bcftools stats output. If not present, the script will use abbreviated source file names for the titles. -v, --vectors Generate vector graphics for PDF images, the opposite of -r, --raster- ize. -T, --main-title STRING Main title for the PDF. Example: # Generate the stats bcftools stats -s - > file.vchk # Plot the stats plot-vcfstats -p outdir file.vchk # The final looks can be customized by editing the generated # 'outdir/plot.py' script and re-running manually cd outdir && python plot.py && pdflatex summary.tex roh-viz [OPTIONS] Script for visualizing the output of bcftools roh. It creates a HTML/JavaScript document which can be interactively viewed in your favorite web browser. -i, --RoH-file FILE Output of bcftools/roh -l, --min-length NUM Mimimum length of ROH [1e6] -o, --output FILE HTML output file -r, --regions LIST List of chromosomes/regions -s, --samples LIST List of samples to show -v, --VCF-file FILE VCF file to determine homozygosity rate -h, -?, --help This help message Example: # Find RoH regions bcftools roh --AF-dflt 0.5 -G 30 -Or -o roh.txt file.bcf # Create HTML/JavaScript visualization roh-viz -r roh.txt -v file.bcf -o output.html FILTERING EXPRESSIONS These filtering expressions are accepted by most of the commands. Valid expressions may contain: * numerical constants, string constants, file names (indicated by the prefix @) 1, 1.0, 1e-4 "String" @file_name * arithmetic operators (addition, multiplication, subtraction, division, modulo) +, *, -, /, % * comparison operators == (same as =), >, >=, <=, <, != * regex operators "\~" and its negation "!~". The expressions are case sensitive unless "/i" is added. INFO/HAYSTACK ~ "needle" INFO/HAYSTACK ~ "NEEDless/i" * parentheses (, ) * logical operators. See also the examples below and the filtering tutor- ial <http://samtools.github.io/bcftools/howtos/filtering.html> about the distinction between "&&" vs "&" and "||" vs "|". &&, &, ||, | * INFO tags, FORMAT tags, column names INFO/DP or DP FORMAT/DV, FMT/DV, or DV FILTER, QUAL, ID, CHROM, POS, REF, ALT[0] * the FILTER column can be queried as follows: FILTER="PASS" FILTER="." FILTER="A" .. exact match, for example "A;B" does not pass FILTER="A;B" .. exact match, "A;B" and "B;A" pass, everything else fails FILTER!="A" .. exact match, for example "A;B" does pass FILTER~"A" .. subset match, for example both "A" and "A;B" pass FILTER~"A;B" .. subset match, pass only if both "A" and "B" are present FILTER!~"A" .. complement match, for example both "A" and "A;B" fail FILTER!~"A;B" .. complement match, fail if both "A" and "B" are present * 1 (or 0) to test the presence (or absence) of a flag FlagA=1 && FlagB=0 * "." to test missing values DP=".", DP!=".", ALT="." * missing genotypes can be matched regardless of phase and ploidy (".|.", "./.", ".", "0|.") using these expressions GT="mis", GT~"\.", GT!~"\." * missing genotypes can be matched including the phase and ploidy (".|.", "./.", ".") using these expressions GT=".|.", GT="./.", GT="." * sample genotype: reference (haploid or diploid), alternate (hom or het, haploid or diploid), missing genotype, homozygous, heterozygous, hap- loid, ref-ref hom, alt-alt hom, ref-alt het, alt-alt het, haploid ref, haploid alt (case-insensitive) GT="ref" GT="alt" GT="mis" GT="hom" GT="het" GT="hap" GT="RR" GT="AA" GT="RA" or GT="AR" GT="Aa" or GT="aA" GT="R" GT="A" * TYPE for variant type in REF,ALT columns (in- del,snp,mnp,ref,bnd,other,overlap, see TERMINOLOGY). Use the regex op- erator "\~" to require at least one allele of the given type or the equal sign "=" to require that all alleles are of the given type. Com- pare TYPE="snp" TYPE~"snp" TYPE!="snp" TYPE!~"snp" * array subscripts (0-based), "*" for any element, "-" to indicate a range. Note that for querying FORMAT vectors, the colon ":" can be used to select a sample and an element of the vector, as shown in the exam- ples below. INFO/AF[0] > 0.3 .. first AF value bigger than 0.3 FORMAT/AD[0:0] > 30 .. first AD value of the first sample bigger than 30 FORMAT/AD[0:1] .. first sample, second AD value FORMAT/AD[1:0] .. second sample, first AD value DP4[*] == 0 .. any DP4 value FORMAT/DP[0] > 30 .. DP of the first sample bigger than 30 FORMAT/DP[1-3] > 10 .. samples 2-4 FORMAT/DP[1-] < 7 .. all samples but the first FORMAT/DP[0,2-4] > 20 .. samples 1, 3-5 FORMAT/AD[0:1] .. first sample, second AD field FORMAT/AD[0:*], AD[0:] or AD[0] .. first sample, any AD field FORMAT/AD[*:1] or AD[:1] .. any sample, second AD field (DP4[0]+DP4[1])/(DP4[2]+DP4[3]) > 0.3 CSQ[*] ~ "missense_variant.*deleterious" * in addition to array subscripts shown above, it is possible to sub- script arrays of Number=R tags by alleles found in FORMAT/GT (starting with version 1.17). For example FORMAT/AD[GT] > 10 .. require support of more than 10 reads for each allele FORMAT/AD[0:GT] > 10 .. same as above, but in the first sample sSUM(FORMAT/AD[GT]) > 20 .. require total sample depth bigger than 20 * with many samples it can be more practical to provide a file with sam- ple names, one sample name per line GT[@samples.txt]="het" & binom(AD)<0.01 * function on FORMAT tags (over samples) and INFO tags (over vector fields): maximum; minimum; arithmetic mean (AVG is synonymous with MEAN); median; standard deviation from mean; sum; string length; ab- solute value; number of elements: MAX, MIN, AVG, MEAN, MEDIAN, STDEV, SUM, STRLEN, ABS, COUNT Note that functions above evaluate to a single value across all samples and are intended to select sites, not samples, even when applied on FORMAT tags. However, when prefixed with SMPL_ (or "s" for brevity, e.g. SMPL_MAX or sMAX), they will evaluate to a vector of per-sample values when applied on FORMAT tags: SMPL_MAX, SMPL_MIN, SMPL_AVG, SMPL_MEAN, SMPL_MEDIAN, SMPL_STDEV, SMPL_SUM, SMPL_COUNT, sMAX, sMIN, sAVG, sMEAN, sMEDIAN, sSTDEV, sSUM, sCOUNT * two-tailed binomial and fisher test. Note that for N=0 the test evalu- ates to a missing value and when FORMAT/GT is used to determine the vector indices, it evaluates to 1 for homozygous genotypes. binom(FMT/AD) .. GT can be used to determine the correct index binom(AD[0],AD[1]) .. or the fields can be given explicitly phred(binom()) .. the same as binom but phred-scaled fisher(INFO/DP4) fisher(FORMAT/DP4) fisher(FMT/ADF,FMT/ADR) .. GT can be used to determine the correct indices fisher(FMT/ADF[:0,1],FMT/ADR[:0,1]) .. or the fields can be given explicitly * variables calculated on the fly if not present: number of alternate al- leles; number of samples; count of alternate alleles; minor allele count (similar to AC but always picks the allele with frequency smaller than 0.5); frequency of alternate alleles (AF=AC/AN); frequency of mi- nor alleles (MAF=MAC/AN); number of alleles in called genotypes; number of samples with missing genotype; fraction of samples with missing genotype; indel length (deletions negative, insertions positive, bal- anced substitutions zero) N_ALT, N_SAMPLES, AC, MAC, AF, MAF, AN, N_MISSING, F_MISSING, ILEN * the number (N_PASS) or fraction (F_PASS) of samples which pass the ex- pression N_PASS(GQ>90 & GT!="mis") > 90 F_PASS(GQ>90 & GT!="mis") > 0.9 * custom perl filtering. Note that this command is not compiled in by de- fault, see the section Optional Compilation with Perl in the INSTALL file for help and misc/demo-flt.pl for a working example. The demo de- fined the perl subroutine "severity" which can be invoked from the com- mand line as follows: perl:path/to/script.pl; perl.severity(INFO/CSQ) > 3 Notes: * String comparisons and regular expressions are case-insensitive * Comma in strings is interpreted as a separator and when multiple values are compared, the OR logic is used. Consequently, the following two ex- pressions are equivalent but not the third: -i 'TAG="hello,world"' -i 'TAG="hello" || TAG="world"' -i 'TAG="hello" && TAG="world"' * Variables and function names are case-insensitive, but not tag names. For example, "qual" can be used instead of "QUAL", "strlen()" instead of "STRLEN()" , but not "dp" instead of "DP". * When querying multiple values, all elements are tested and the OR logic is used on the result. For example, when querying "TAG=1,2,3,4", it will be evaluated as follows: -i 'TAG[*]=1' .. true, the record will be printed -i 'TAG[*]!=1' .. true -e 'TAG[*]=1' .. false, the record will be discarded -e 'TAG[*]!=1' .. false -i 'TAG[0]=1' .. true -i 'TAG[0]!=1' .. false -e 'TAG[0]=1' .. false -e 'TAG[0]!=1' .. true * When arithmetic operators are used on vectors A and B, the following logic is used to compute the resulting vector C: * C_i = A_i + B_i when length(A)==B(A) and sets length(C)=length(A) * C_i = A_i + B_0 when length(B)=1 and sets length(C)=length(A) * C_i = A_0 + B_i when length(A)=1 and sets length(C)=length(B) * throw an error when length(A)!=length(B) AND length(A)!=1 AND length(B)!=1 Examples: MIN(DV)>5 .. selects the whole site, evaluates min across all values and samples SMPL_MIN(DV)>5 .. selects matching samples, evaluates within samples MIN(DV/DP)>0.3 MIN(DP)>10 & MIN(DV)>3 FMT/DP>10 & FMT/GQ>10 .. both conditions must be satisfied within one sample FMT/DP>10 && FMT/GQ>10 .. the conditions can be satisfied in different samples QUAL>10 | FMT/GQ>10 .. true for sites with QUAL>10 or a sample with GQ>10, but selects only samples with GQ>10 QUAL>10 || FMT/GQ>10 .. true for sites with QUAL>10 or a sample with GQ>10, plus selects all samples at such sites TYPE="snp" && QUAL>=10 && (DP4[2]+DP4[3] > 2) COUNT(GT="hom")=0 .. no homozygous genotypes at the site AVG(GQ)>50 .. average (arithmetic mean) of genotype qualities bigger than 50 ID=@file .. selects lines with ID present in the file ID!=@~/file .. skip lines with ID present in the ~/file INFO/TAG=@file .. selects lines with INFO/TAG value present in the file MAF[0]<0.05 .. select rare variants at 5% cutoff POS>=100 .. restrict your range query, e.g. 20:100-200 to strictly sites with POS in that range. Shell expansion: Note that expressions must often be quoted because some characters have special meaning in the shell. An example of expression enclosed in single quotes which cause that the whole expression is passed to the program as intended: bcftools view -i 'ID!="." & MAF[0]<0.01' Please refer to the documentation of your shell for details. TERMINOLOGY The program and the documentation uses the following terminology, multiple terms can be used interchangeably for the same VCF record type REF ALT --------- C . .. reference allele / non-variant site / ref-only site C T .. SNP or SNV (single-nucleotide polymorphism or variant), used interchangeably CC TT .. MNP (multi-nucleotide polymorphism) CAAA C .. indel, deletion (regardless of length) C CAAA .. indel, insertion (regardless of length) C <*> .. gVCF block, the allele <*> is a placeholder for alternate allele possibly missed because of low coverage C <NON_REF> .. synonymous to <*> C * .. overlapping deletion C <INS> .. symbolic allele, known also as 'other [than above]' PERFORMANCE HTSlib was designed with BCF format in mind. When parsing VCF files, all records are internally converted into BCF representation. Simple opera- tions, like removing a single column from a VCF file, can be therefore done much faster with standard UNIX commands, such as awk or cut. Therefore it is recommended to use BCF as input/output format whenever possible to avoid large overhead of the VCF -> BCF -> VCF conversion. BUGS Please report any bugs you encounter on the github website: http://github.com/samtools/bcftools AUTHORS Heng Li from the Sanger Institute wrote the original C version of htslib, samtools and bcftools. Bob Handsaker from the Broad Institute implemented the BGZF library. Petr Danecek is maintaining and further developing bcftools, together with the rest of the samtools team <https://www.sanger.ac.uk/tool/samtools-bcftools-htslib>. Many other people contributed to the program and to the file format specifications, both di- rectly and indirectly by providing patches, testing and reporting bugs. We thank them all. RESOURCES BCFtools GitHub website: http://github.com/samtools/bcftools Samtools GitHub website: http://github.com/samtools/samtools HTSlib GitHub website: http://github.com/samtools/htslib File format specifications: http://samtools.github.io/hts-specs BCFtools documentation: http://samtools.github.io/bcftools BCFtools wiki page: https://github.com/samtools/bcftools/wiki COPYING The MIT/Expat License or GPL License, see the LICENSE document for details. Copyright (c) Genome Research Ltd. 2026-03-18 BCFTOOLS(1)
NAME | SYNOPSIS | DESCRIPTION | LIST OF COMMANDS | LIST OF SCRIPTS | COMMANDS AND OPTIONS | SCRIPTS | FILTERING EXPRESSIONS | TERMINOLOGY | PERFORMANCE | BUGS | AUTHORS | RESOURCES | COPYING
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