'\" t .TH samtools-markdup 1 "21 February 2023" "samtools-1.17" "Bioinformatics tools" .SH NAME samtools-markdup \- mark duplicate alignments in a coordinate sorted file .\" .\" Copyright (C) 2008-2011, 2013-2022 Genome Research Ltd. .\" Portions copyright (C) 2010, 2011 Broad Institute. .\" .\" Author: Heng Li .\" Author: Joshua C. Randall .\" .\" Permission is hereby granted, free of charge, to any person obtaining a .\" copy of this software and associated documentation files (the "Software"), .\" to deal in the Software without restriction, including without limitation .\" the rights to use, copy, modify, merge, publish, distribute, sublicense, .\" and/or sell copies of the Software, and to permit persons to whom the .\" Software is furnished to do so, subject to the following conditions: .\" .\" The above copyright notice and this permission notice shall be included in .\" all copies or substantial portions of the Software. .\" .\" THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR .\" IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, .\" FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL .\" THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER .\" LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING .\" FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER .\" DEALINGS IN THE SOFTWARE. . .\" For code blocks and examples (cf groff's Ultrix-specific man macros) .de EX . in +\\$1 . nf . ft CR .. .de EE . ft . fi . in .. . .SH SYNOPSIS .PP samtools markdup .RB [ -l .IR length ] .RB [ -r ] .RB [ -T ] .RB [ -S ] .RB [ -s ] .RB [ -f .IR file ] .RB [ --json ] .RB [ -d .IR distance ] .RB [ -c ] .RB [ -t ] .RB [ -m ] .RB [ --mode ] .RB [ --include-fails ] .RB [ --no-PG ] .RB [ -u ] .RB [ --no-multi-dup ] .RB [ --read-coords ] .RB [ --coords-order ] .RB [ --barcode-tag ] .RB [ --barcode-name ] .RB [ --barcode-rgx ] .RB [ --use-read-groups ] .I in.algsort.bam out.bam .SH DESCRIPTION .PP Mark duplicate alignments from a coordinate sorted file that has been run through \fBsamtools fixmate\fR with the \fB-m\fR option. This program relies on the MC and ms tags that fixmate provides. .SH OPTIONS .TP 11 .BI "-l " INT .RI "Expected maximum read length of " INT " bases." [300] .TP .B -r Remove duplicate reads. .TP .BI "-T " PREFIX Write temporary files to .IB PREFIX . samtools . nnnn . mmmm . tmp .TP .B -S Mark supplementary reads of duplicates as duplicates. .TP .B -s Print some basic stats. See STATISTICS. .TP .BI "-f " file Write stats to named file. .TP .B --json Output stats in JSON format. .TP .BI "-d " distance The optical duplicate distance. Suggested settings of 100 for HiSeq style platforms or about 2500 for NovaSeq ones. Default is 0 to not look for optical duplicates. When set, duplicate reads are tagged with \fBdt:Z:SQ\fR for optical duplicates and \fBdt:Z:LB\fR otherwise. Calculation of distance depends on coordinate data embedded in the read names produced by the Illumina sequencing machines. Optical duplicate detection will not work on non standard names without the use of \fB--read-coords\fR. .TP .B -c Clear previous duplicate settings and tags. .TP .B -t Mark duplicates with the name of the original in a \fBdo\fR tag. .TP .BI "-m, --mode " TYPE Duplicate decision method for paired reads. Values are \fBt\fR or \fBs\fR. Mode \fBt\fR measures positions based on template start/end (default). Mode \fBs\fR measures positions based on sequence start. While the two methods identify mostly the same reads as duplicates, mode \fBs\fR tends to return more results. Unpaired reads are treated identically by both modes. .TP .B -u Output uncompressed SAM, BAM or CRAM. .TP .B --include-fails Include quality checked failed reads. .TP .B --no-multi-dup Stop checking duplicates of duplicates for correctness. While still marking reads as duplicates further checks to make sure all optical duplicates are found are not carried out. Also operates on \fB-t\fR tagging where reads may tagged with a better quality read but not necessarily the best one. Using this option can speed up duplicate marking when there are a great many duplicates for each original read. .TP .BI "--read-coords " REGEX This takes a POSIX regular expression for at least x and y to be used in optical duplicate marking It can also include another part of the read name to test for equality, eg lane:tile elements. Elements wanted are captured with parentheses. Examples below. .TP .BI "--coords-order " ORDER The order of the elements captured in the regular expression. Default is txy where t is a part of the read name selected for string comparison and x/y the coordinates used for optical duplicate detection. Valid orders are: txy, tyx, xyt, yxt, xty, ytx, xy and yx. .TP .BI "--barcode-tag " TAG Duplicates must now also match the barcode tag. .TP .B --barcode-name Use the UMI/barcode embedded in the read name (eigth colon delimited part). .TP .BI "--barcode-rgx " REGEX Regex for barcode in the readname (alternative to --barcode-name). .TP .B --use-read-groups The @RG tags must now also match to be a duplicate. .TP .B --no-PG Do not add a PG line to the output file. .TP .BI "-@, --threads " INT Number of input/output compression threads to use in addition to main thread [0]. .SH STATISTICS Entries are: .br \fBCOMMAND\fR: the command line. .br \fBREAD\fR: number of reads read in. .br \fBWRITTEN\fR: reads written out. .br \fBEXCLUDED\fR: reads ignored. See below. .br \fBEXAMINED\fR: reads examined for duplication. .br \fBPAIRED\fR: reads that are part of a pair. .br \fBSINGLE\fR: reads that are not part of a pair. .br \fBDUPLICATE PAIR\fR: reads in a duplicate pair. .br \fBDUPLICATE SINGLE\fR: single read duplicates. .br \fBDUPLICATE PAIR OPTICAL\fR: optical duplicate paired reads. .br \fBDUPLICATE SINGLE OPTICAL\fR: optical duplicate single reads. .br \fBDUPLICATE NON PRIMARY\fR: supplementary/secondary duplicate reads. .br \fBDUPLICATE NON PRIMARY OPTICAL\fR: supplementary/secondary optical duplicate reads. .br \fBDUPLICATE PRIMARY TOTAL\fR: number of primary duplicate reads. .br \fBDUPLICATE TOTAL\fR: total number of duplicate reads. .br \fBESTIMATED LIBRARY SIZE\fR: estimate of the number of unique fragments in the sequencing library. Estimated library size makes various assumptions e.g. the library consists of unique fragments that are randomly selected (with replacement) with equal probability. This is unlikely to be true in practice. However it can provide a useful guide into how many unique read pairs are likely to be available. In particular it can be used to determine how much more data might be obtained by further sequencing of the library. Excluded reads are those marked as secondary, supplementary or unmapped. By default QC failed reads are also excluded but can be included as an option. Excluded reads are not used for calculating duplicates. They can optionally be marked as duplicates if they have a primary that is also a duplicate. .SH EXAMPLES This first collate command can be omitted if the file is already name ordered or collated: .EX 4 samtools collate -o namecollate.bam example.bam .EE Add ms and MC tags for markdup to use later: .EX 4 samtools fixmate -m namecollate.bam fixmate.bam .EE Markdup needs position order: .EX 4 samtools sort -o positionsort.bam fixmate.bam .EE Finally mark duplicates: .EX 4 samtools markdup positionsort.bam markdup.bam .EE Typically the fixmate step would be applied immediately after sequence alignment and the markdup step after sorting by chromosome and position. Thus no \fIadditional\fR sort steps are normally needed. To use the regex to obtain coordinates from reads, two or three values have to be captured. To mimic the normal behaviour and match a read name of the format \fImachine:run:flowcell:lane:tile:x:y\fR use: .EX 4 --read-coords '([!-9;-?A-~]+:[0-9]+:[0-9]+:[0-9]+:[0-9]+):([0-9]+):([0-9]+)' --coords-order txy .EE To match only the coordinates of \fIx:y:randomstuff\fR use: .EX 4 --read-coords '^([[:digit:]]+):([[:digit:]]+)' --coords-order xy .EE To use a barcode from the read name matching the Illumina example of \fINDX550136:7:H2MTNBDXX:1:13302:3141:10799:AAGGATG+TCGGAGA\fR use: .EX 4 --barcode-rgx '[0-9A-Za-z]+:[0-9]+:[0-9A-Za-z]+:[0-9]+:[0-9]+:[0-9]+:[0-9]+:([!-?A-~]+)' .EE It is possible that complex regular expressions may slow the running of the program. It would be best to keep them simple. .SH AUTHOR .PP Written by Andrew Whitwham from the Sanger Institute. .SH SEE ALSO .IR samtools (1), .IR samtools-sort (1), .IR samtools-collate (1), .IR samtools-fixmate (1) .PP Samtools website: