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samtools-consensus(1) Bioinformatics tools samtools-consensus(1) NAME samtools consensus - produces a consensus FASTA/FASTQ/PILEUP SYNOPSIS samtools consensus [-saAMq] [-r region] [-f format] [-l line-len] [-d min-depth] [-C cutoff] [-c call-fract] [-H het-fract] in.bam DESCRIPTION Generate consensus from a SAM, BAM or CRAM file based on the contents of the alignment records. The consensus is written either as FASTA, FASTQ, or a pileup oriented format. This is selected using the -f FOR- MAT option. The default output for FASTA and FASTQ formats include one base per non-gap consensus. Hence insertions with respect to the aligned refer- ence will be included and deletions removed. This behaviour can be controlled with the --show-ins and --show-del options. This could be used to compute a new reference from sequences assemblies to realign against. The pileup-style format strictly adheres to one row per consensus loca- tion, differing from the one row per reference based used in the re- lated "samtools mpileup" command. This means the base quality values for inserted columns are reported. The base quality value of gaps (ei- ther within an insertion or otherwise) are determined as the average of the surrounding non-gap bases. The columns shown are the reference name, position, nth base at that position (zero if not an insertion), consensus call, consensus confidence, sequences and quality values. Two consensus calling algorithms are offered. The default computes a heterozygous consensus in a Bayesian manner, derived from the "Gap5" consensus algorithm. Quality values are also tweaked to take into ac- count other nearby low quality values. This can also be disabled, us- ing the --no-adj-qual option. This method also utilises the mapping qualities, unless the --no-use-MQ option is used. Mapping qualities are also auto-scaled to take into account the local reference variation by processing the MD:Z tag, un- less --no-adj-MQ is used. Mapping qualities can be capped between a minimum (--low-MQ) and maximum (--high-MQ), although the defaults are liberal and trust the data to be true. Finally an overall scale on the resulting mapping quality can be supplied (--scale-MQ, defaulting to 1.0). This has the effect of favouring more calls with a higher false positive rate (values greater than 1.0) or being more cautious with higher false negative rates and lower false positive (values less than 1.0). The second method is a simple frequency counting algorithm, summing ei- ther +1 for each base type or +qual if the --use-qual option is speci- fied. This is enabled with the --mode simple option. The summed share of a specific base type is then compared against the total possible and if this is above the --call-fract fraction parameter then the most likely base type is called, or "N" otherwise (or absent if it is a gap). The --ambig option permits generation of ambiguity codes instead of "N", provided the minimum fraction of the second most common base type to the most common is above the --het-fract fraction. OPTIONS General options that apply to both algorithms: -r REG, --region REG Limit the query to region REG. This requires an index. -f FMT, --format FMT Produce format FMT, with "fastq", "fasta" and "pileup" as permitted options. -l N, --line-len N Sets the maximum line length of line-wrapped fasta and fastq formats to N. -o FILE, --output FILE Output consensus to FILE instead of stdout. -m STR, --mode STR Select the consensus algorithm. Valid modes are "simple" frequency counting and the "bayesian" (Gap5) methods, with Bayesian being the default. (Note case does not matter, so "Bayesian" is accepted too.) There are a variety of bayesian methods. Straight "bayesian" is the best set suitable for the other parameters selected. The choice of internal para- meters may change depending on the "--P-indel" score. This method distinguishes between substitution and indel error rates. The old Samtools consensus in version 1.16 did not distinguish types of errors, but for compatibility the "bayesian_116" mode may be selected to replicate this. -a Outputs all bases, from start to end of reference, even when the aligned data does not extend to the ends. This is most useful for construction of a full length reference sequence. -a -a, -aa Output absolutely all positions, including references with no data aligned against them. --rf, --incl-flags STR|INT Only include reads with at least one FLAG bit set. Defaults to zero, which filters no reads. --ff, --excl-flags STR|INT Exclude reads with any FLAG bit set. Defaults to "UNMAP,SEC- ONDARY,QCFAIL,DUP". --min-MQ INT Filters out reads with a mapping quality below INT. This de- faults to zero. --min-BQ INT Filters out bases with a base quality below INT. This de- faults to zero. --show-del yes/no Whether to show deletions as "*" (yes) or to omit from the output (no). Defaults to no. --show-ins yes/no Whether to show insertions in the consensus. Defaults to yes. --mark-ins Insertions, when shown, are normally recorded in the consen- sus with plain 7-bit ASCII (ACGT, or acgt if heterozygous). However this makes it impossible to identify the mapping be- tween consensus coordinates and the original reference coor- dinates. If fasta output is selected then the option adds an underscore before every inserted base, plus a corresponding character in the quality for fastq format. When used in con- junction with -a --show-del yes, this permits an easy deriva- tion of the consensus to reference coordinate mapping. -A, --ambig Enables IUPAC ambiguity codes in the consensus output. With- out this the output will be limited to A, C, G, T, N and *. -d D, --min-depth D The minimum depth required to make a call. Defaults to 1. Failing this depth check will produce consensus "N", or ab- sent if it is an insertion. Note this check is performed af- ter filtering by flags and mapping/base quality. The following options apply only to the simple consensus mode: -q, --use-qual For the simple consensus algorithm, this enables use of base quality values. Instead of summing 1 per base called, it sums the base quality instead. These sums are also used in the --call-fract and --het-fract parameters too. Quality values are always used for the "Gap5" consensus method and this option has no affect. Note currently quality values only affect SNPs and not inserted sequences, which still get scores with a fixed +1 per base type occurrence. -H H, --het-fract H For consensus columns containing multiple base types, if the second most frequent type is at least H fraction of the most common type then a heterozygous base type will be reported in the consensus. Otherwise the most common base is used, pro- vided it meets the --call-fract parameter (otherwise "N"). The fractions computed may be modified by the use of quality values if the -q option is enabled. Note although IUPAC has ambiguity codes for A,C,G,T vs any other A,C,G,T it does not have codes for A,C,G,T vs gap (such as in a heterozygous deletion). Given the lack of any official code, we use lower-case letter to symbolise a half-present base type. -c C, --call-fract C Only used for the simple consensus algorithm. Require at least C fraction of bases agreeing with the most likely con- sensus call to emit that base type. This defaults to 0.75. Failing this check will output "N". The following options apply only to Bayesian consensus mode enabled (default on). -C C, --cutoff C Only used for the Gap5 consensus mode, which produces a Phred style score for the final consensus quality. If this is be- low C then the consensus is called as "N". --use-MQ, --no-use-MQ Enable or disable the use of mapping qualities. Defaults to on. --adj-MQ, --no-adj-MQ If mapping qualities are used, this controls whether they are scaled by the local number of mismatches to the reference. The reference is unknown by this tool, so this data is ob- tained from the MD:Z auxiliary tag (or ignored if not present). Defaults to on. --NM-halo INT Specifies the distance either side of the base call being considered for computing the number of local mismatches. --low-MQ MIN, --high-MQ MAX Specifies a minimum and maximum value of the mapping quality. These are not filters and instead simply put upper and lower caps on the values. The defaults are 0 and 60. --scale-MQ FLOAT This is a general multiplicative mapping quality scaling factor. The effect is to globally raise or lower the quality values used in the consensus algorithm. Defaults to 1.0, which leaves the values unchanged. --P-het FLOAT Controls the likelihood of any position being a heterozygous site. This is used in the priors for the Bayesian calcula- tions, and has little difference on deep data. Defaults to 1e-3. Smaller numbers makes the algorithm more likely to call a pure base type. Note the algorithm will always com- pute the probability of the base being homozygous vs het- erozygous, irrespective of whether the output is reported as ambiguous (it will be "N" if deemed to be heterozygous with- out --ambig mode enabled). --P-indel FLOAT Controls the likelihood of small indels. This is used in the priors for the Bayesian calculations, and has little differ- ence on deep data. Defaults to 2e-4. --het-scale FLOAT This is a multiplicative correction applied per base quality before adding to the heterozygous hypotheses. Reducing it means fewer heterozygous calls are made. This oftens leads a significant reduction in false positive het calls, for some increase in false negatives (mislabelling real heterozygous sites as homozygous). It is usually beneficial to reduce this on instruments where a significant proportion of bases may be aligned in the wrong column due to insertions and deletions leading to alignment errors and reference bias. It can be considered as a het sensitivity tuning parameter. De- faults to 1.0 (nop). -p, --homopoly-fix Some technologies that call runs of the same base type to- gether always put the lowest quality calls at one end. This can cause problems when reverse complementing and comparing alignments with indels. This option averages the qualities at both ends to avoid orientation biases. Recommended for old 454 or PacBio HiFi data sets. --homopoly-score FLOAT The -p option also reduces confidence values within homopoly- mers due to an additional likelihood of sequence specific er- rors. The quality values are multiplied by FLOAT. This de- faults to 0.5, but is not used if -p was not specified. Ad- justing this score also automatically enables -p. -t, --qual-calibration FILE Loads a quality calibration table from FILE. The format of this is a series of lines with the following fields, each starting with the literal text "QUAL": QUAL value substitution undercall overcall Lines starting with a "#" are ignored. Each line maps a recorded quality value to the Phred equivalent score for sub- stitution, undercall and overcall errors. Quality values are expected to be sorted in increasing numerical order, but may skip values. This allows the consensus algorithm to know the most likely cause of an error, and whether the instrument is more likely to have indel errors (more common in some long read technologies) or substitution errors (more common in clocked short-read instruments). Some pre-defined calibration tables are built in. These are specified with a fake filename starting with a colon. See the -X option for more details. Note due to the additional heuristics applied by the consen- sus algorithm, these recalibration tables are not a true re- flection of the instrument error rates and are a work in progress. -X, --config STR Specifies predefined sets of configuration parameters. Ac- ceptable values for STR are defined below, along with the list of parameters they are equivalent to. hiseq --qual-calibration :hiseq hifi --qual-calibration :hifi --homopoly-fix 0.3 --low- MQ 5 --scale-MQ 1.5 --het-scale 0.37 r10.4_sup --qual-calibration :r10.4_sup --homopoly-fix 0.3 --low-MQ 5 --scale-MQ 1.5 --het-scale 0.37 r10.4_dup --qual-calibration :r10.4_dup --homopoly-fix 0.3 --low-MQ 5 --scale-MQ 1.5 --het-scale 0.37 ultima --qual-calibration :ultima --homopoly-fix 0.3 --low-MQ 10 --scale-MQ 2 --het-scale 0.37 EXAMPLES - Create a modified FASTA reference that has a 1:1 coordinate cor- respondence with the original reference used in alignment. samtools consensus -a --show-ins no in.bam -o ref.fa - Create a FASTQ file for the contigs with aligned data, including insertions. samtools consensus -f fastq in.bam -o cons.fq AUTHOR Written by James Bonfield from the Sanger Institute. SEE ALSO samtools(1), samtools-mpileup(1), Samtools website: <http://www.htslib.org/> samtools-1.21 12 September 2024 samtools-consensus(1)
NAME | SYNOPSIS | DESCRIPTION | OPTIONS | EXAMPLES | AUTHOR | SEE ALSO
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