Written by: Greg Faust (gf4ea@virginia.edu) Ira Hall Lab, University of Virginia
Also see: SAMBLASTER_Supplemental.pdf for additonal discussion and statistics about the duplicates marked by samblaster vs. Picard using the NA12878 sample dataset. Click the preceeding link or download the file from this repository.
Current version: 0.1.26
Support for Linux and OSX (Version 10.7 or higher).
samblaster is a fast and flexible program for marking duplicates in read-id grouped1 paired-end SAM files. It can also optionally output discordant read pairs and/or split read mappings to separate SAM files, and/or unmapped/clipped reads to a separate FASTQ file. When marking duplicates, samblaster will require approximately 20MB of memory per 1M read pairs.
samblaster is self contained and therefore has no installation dependencies beyond g++ and make.
samblaster can be downloaded from the releases tab or manually downloaded via git clone. Afterwards, simply use make and copy samblaster to a directory in your path. For example:
git clone git://github.com/GregoryFaust/samblaster.git
cd samblaster
make
cp samblaster /usr/local/bin/.
See the SAM File Format Specification for details about the SAM alignment format.
By default, samblaster reads SAM input from stdin and writes SAM to stdout. Input SAM files usually contain paired end data (see Duplicate Identification below), must contain a sequence header, and must be read-id grouped1. By default, the output SAM file will contain all the alignments in the same order as the input, with duplicates marked with SAM FLAG 0x400. The --removeDups option will instead remove duplicate alignments from the output file.
1A read-id grouped SAM file is one in which all alignments for a read-id (QNAME) are grouped together in adjacent lines. Aligners naturally produce such files. They can also be created by sorting a SAM file by read-id. But as shown below, sorting the input to samblaster by read-id is not required if the alignments are already grouped.
COMMON USAGE SCENARIOS:
To take input alignments directly from bwa mem and output to samtools view to compress SAM to BAM:
bwa mem <idxbase> samp.r1.fq samp.r2.fq | samblaster | samtools view -Sb - > samp.out.bam
When using the bwa mem -M option, also use the samblaster -M option:
bwa mem -M <idxbase> samp.r1.fq samp.r2.fq | samblaster -M | samtools view -Sb - > samp.out.bam
To additionally output discordant read pairs and split read alignments:
bwa mem <idxbase> samp.r1.fq samp.r2.fq | samblaster -e -d samp.disc.sam -s samp.split.sam | samtools view -Sb - > samp.out.bam
To pull split reads and discordants read pairs from a pre-existing BAM file with duplicates already marked:
samtools view -h samp.bam | samblaster -a -e -d samp.disc.sam -s samp.split.sam -o /dev/null
To process a BAM file of singleton long reads to pull split and/or unmapped reads with/without marking duplicates:
samtools view -h samp.bam | samblaster --ignoreUnmated [-e] --maxReadLength 100000 [-s samp.split.sam] [-u samp.umc.fasta] | samtools view -Sb - > samp.out.bam
samtools view -h samp.bam | samblaster --ignoreUnmated -a [-e] [-s samp.split.sam] [-u samp.umc.fasta] -o /dev/null
OPTIONS: Default values enclosed in square brackets []
Input/Output Options:
-i --input FILE Input sam file [stdin].
-o --output FILE Output sam file for all input alignments [stdout].
-d --discordantFile FILE Output discordant read pairs to this file. [no discordant file output]
-s --splitterFile FILE Output split reads to this file abiding by paramaters below. [no splitter file output]
-u --unmappedFile FILE Output unmapped/clipped reads as FASTQ to this file abiding by parameters below. [no unmapped file output].
Requires soft clipping in input file. Will output FASTQ if QUAL information available, otherwise FASTA.
Other Options:
-a --acceptDupMarks Accept duplicate marks already in input file instead of looking for duplicates in the input.
-e --excludeDups Exclude reads marked as duplicates from discordant, splitter, and/or unmapped file.
-r --removeDups Remove duplicates reads from all output files. (Implies --excludeDups).
--addMateTags Add MC and MQ tags to all output paired-end SAM lines.
--ignoreUnmated Suppress abort on unmated alignments. Use only when sure input is read-id grouped,
and either paired-end alignments have been filtered or the input file contains singleton reads.
--ignoreUnmated is not recommended for general use on paired-end data. It disables checks that detect incorrectly sorted input.
-M Compatibility mode (details below); both FLAG 0x100 and 0x800 denote supplementary (chimeric). Similar to bwa mem -M option.
--maxReadLength INT Maximum allowed length of the SEQ/QUAL string in the input file. [500]
--maxSplitCount INT Maximum number of split alignments for a read to be included in splitter file. [2]
--maxUnmappedBases INT Maximum number of un-aligned bases between two alignments to be included in splitter file. [50]
--minIndelSize INT Minimum structural variant feature size for split alignments to be included in splitter file. [50]
--minNonOverlap INT Minimum non-overlaping base pairs between two alignments for a read to be included in splitter file. [20]
--minClipSize INT Minumum number of bases a mapped read must be clipped to be included in unmapped file. [20]
-h --help Print samblaster help to stderr.
-q --quiet Output fewer statistics.
--version Print samblaster version number to stderr.
ALIGNMENT TYPE DEFINITIONS: Below, we will use the following definitions for alignment types. Starting with samblaster release 0.1.22, these definitions are affected by the use of the -M option. By default, samblaster will use the current definitions of alignment types as specified in the SAM Specification. Namely, alignments marked with FLAG 0x100 are considered secondary, while those marked with FLAG 0x800 are considered supplementary. If the -M option is specified, alignments marked with either FLAG 0x100 or 0x800 are considered supplementary, and no alignments are considered secondary. A primary alignment is always one that is neither secondary nor supplementary. Only primary and supplementary alignments are used to find chimeric (split-read) mappings. The -M flag is used for backward compatibility with older SAM/BAM files in which "chimeric" alignments were marked with FLAG 0x100, and should also be used with output from more recent runs of bwa mem using its -M option.
DUPLICATE IDENTIFICATION: A duplicate read pair is defined as a pair that has the same signature for each mapped read as a previous read pair in the input SAM file. The signature is comprised of the combination of the sequence name, strand, and the reference offset where the 5' end of the read would fall if the read were fully aligned (not clipped) at its 5' end. The 5' aligned reference position is calculated using a combination of the POS field, the strand, and the CIGAR string. This definition of signature matches that used by Picard MarkDuplicates.
- For pairs in which both reads are mapped, both signatures must match.
- For pairs in which only one side is mapped (an "orphan"), the signature of the mapped read must match a previously seen orphan. Starting with samblaster 0.1.25, forward and reverse strand orphans/singletons are allowed to be duplicates of each other. In an orphan pair, the unmapped read need not appear in the input file. In addition, mapped non-paired singleton (possible long) read alignments will be treated the same as an orphan pair with a missing unmapped read.
- No doubly unmapped pair will be marked as a duplicate.
- Any secondary or supplementary alignment associated with a duplicate primary alignment will also be marked as a duplicate.
DISCORDANT READ PAIR IDENTIFICATION: A discordant read pair is one which meets all of the following criteria:
- Both side of the read pair are mapped (neither FLAG 0x4 or 0x8 is set).
- The properly paired FLAG (0x2) is not set.
- Secondary or supplementary alignments are never output as discordant, although a discordant read pair can have such alignments associated with them.
- Duplicate read pairs that meet the above criteria will be output as discordant unless the -e option is used.
SPLIT READ IDENTIFICATION: Split Read alignments are derived from a single read when one portion of the read aligns to a different region of the reference genome than another portion of the read. Such pairs of alignments often define a structural variant (SV) breakpoint, and are therefore useful input to SV detection algorithms such as LUMPY. samblaster uses the following strategy to identify split reads alignments.
- Identify reads that have between two and --maxSplitCount primary and supplementary alignments.
- Sort these alignments by their strand-normalized position along the read.
- Two alignments are output as splitters if they are adjacent on the read, and meet these criteria:
- each covers at least --minNonOverlap base pairs of the read that the other does not.
- the two alignments map to different reference sequences and/or strands.
- the two alignments map to the same sequence and strand, and represent a SV that is at least --minIndelSize in length, and have at most --maxUnmappedBases of un-aligned base pairs between them.
- Split read alignments that are part of a duplicate read will be output unless the -e option is used.
UNMAPPED/CLIPPED READ IDENTIFICATION: An unmapped or clipped read is a primary alignment that is unaligned over all or part of its length respectively. The lack of a full alignment may be caused by a SV breakpoint that falls within the read. Therefore, samblaster will optionally output such reads to a FASTQ file for re-alignment by a tool, such as YAHA, geared toward finding split-read mappings. samblaster applies the following strategy to identify and output unmapped/clipped reads:
- An unmapped read has the unmapped read FLAG set (0x4).
- A clipped read is a mapped read with a CIGAR string that begins or ends with at least --minClipSize unaligned bases (CIGAR code S and/or H), and is not from a read that has one or more supplementary alignments.
- In order for samblaster to output the entire sequence for clipped reads, the input SAM file must have soft clipped primary alignments.
- samblaster will output unmapped/clipped reads into a FASTQ file if QUAL information is available in the input file, and a FASTA file if not.
- Unmapped/clipped reads that are part of a duplicate read pair will be output unless the -e option is used.