Filter SAM file for soft and hard clipped alignments

Most short read aligners perform local alignment of reads to the reference genome. Examples includes bwa mem, minimap2, and bowtie2 (unless in --end-to-end mode). This means the ends of the read may not be part of the best alignment.

This can be caused by:

• adapter sequences (aren't in the reference)
• poor quality bases (mismatches only make the alignment score worse)
• structural variation in your sample compared to the reference
• reads overlapping the start and end of contigs (including circular genomes)

Read aligners output a SAM file. Column 6 in this format stores the CIGAR string. which describes which parts of the read aligned and which didn't. The unaligned ends of the read can be "soft" or "hard" clipped, denoted with S and H at each end of the CIGAR string. It is possible for both types to be present, but that is not common. Soft and hard don't mean anything biologically, they just refer to whether the full read sequence is in the SAM file or not.

Some examples of a 100bp aligned read:

• 100M - the read is fully aligned
• 30S70M - soft clipped 30 bp on left end, followed by 70 aligned bp.
• 95M5H - 95 aligned bp, followed by 5 bp unaligned (hard clipped).
• 25S40M35S - only the middle 40bp of the read aligned

One may wish to remove these alignments to avoid downstream problems. In particular, samclip was designed to remove clipped alignments to improve variant calling, by removing suspicious local aligments causing false positives near structural variation. However, it does keep them if they hit the ends of contigs, which is particularly important given the lower coverage often observed at those locations.

samclip has no dependencies except Perl 5.10 or higher. It only uses core modules, so no CPAN needed.

% cd /usr/local/bin  # choose a folder in your $PATH
% wget https://raw.githubusercontent.com/tseemann/samclip/master/samclip
% chmod +x samclip

% brew install brewsci/bio/samclip

% conda install -c bioconda -c conda-forge samclip

% git clone https://github.com/tseemann/samclip.git
% cp samclip/samclip /usr/local/bin # choose a folder in your $PATH

% ./samclip --version
samclip 0.2

% ./samclip --help
SYNOPSIS
  Filter SAM file for soft & hard clipped alignments
AUTHOR
  Torsten Seemann (@torstenseemann)
USAGE
  % samclip --ref ref.fa < in.sam > out.sam
  % minimap2 ref.fa R1.fq R2.fq | samclip --ref ref.fa | samtools sort > out.bam
OPTIONS
  --help         This help
  --version      Print version and exit
  --ref FASTA    Reference genome - needs FASTA.fai index
  --max NUM      Maximum clip length to allow (default=5)
  --invert       Output rejected SAM lines and ignore good ones
  --debug        Print verbose debug info to stderr
  --progress N   Print progress every NUM records (default=100000,none=0)
HOMEPAGE
  https://github.com/tseemann/samclip

% samclip --ref ref.fa < in.sam > out.sam

% samclip --ref ref.fa in1.sam in2.sam > out.sam

# the -h is to emsure the SAM header is included
% samtools view -h in.bam | samclip --ref ref.fa | samtools sort > out.bam

% bwa mem ref.fa R1.fq R2.fq | samclip --ref ref.fa | samtools sort > out.bam 

• --ref FILE should be a FASTA file indexed with samtools faidx FILE
• --max INTEGER is the maximum soft+hard clipping to allow, use 0 to reject all bar contig edges
• --invert will output the records that would have clipped and discard the good ones
• --progress N will print a progress message every N records it processes, use 0 to disable
• --debug is verbose debugging information for testing purposes

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GPL v3

Torsten Seemann