FindSwitchbackReads

Overview

Group: SAM/BAM

Finds reads where a template switch occurred during library construction. Some library construction methods, notably ultra-low-input shotgun methods, are prone to template switching events that create molecules (templates, inserts) that instead of being a linear copy of a segment of genomic DNA, instead are chimeras formed by starting on one strand of DNA and then, switching to the opposite strand. Frequently when the switch occurs there may be a small offset between where the first strand was departed and the opposite strand joined.

Algorithm

Templates that contain strand switch events (switch-backs) are found by this tool in two different ways:

1. By looking at reads that contain soft-clipped bases at their 5’ end that, when reverse complemented, matches the genome proximal to the 5’-most mapped base of the read. We call these matches “read based switchbacks”. Finding read based switchbacks is based on several parameters:

   1. max-offset controls how far away to search for the reverse-complemented sequence. The default value of 35 allows matches to be found when the soft-clipped sequence matches the genome starting at most 35bp from the 5’ mapped position of the read, and reading in the opposite direction.
   2. min-length controls the minimum number of soft-clipped bases that must exist to trigger the search. Given that the search looks at 2 * max-offset locations (default=70) it is important that min-length is set such that 4^min-length >> 2 * max-offset` in order to avoid false positives.
   3. max-error-rate allows for some mismatches to exist between the soft-clipped sequence and the genome when matching.

2. By identifying templates with FF or RR (aka tandem) orientation where it is surmised that the template switch occurred in the un-sequenced region of the template between R1 and R2. We call these tandem based switchbacks. This is controlled by a single parameter, max-gap, which causes the tool to only identify a tandem read pair as a switch-back if the gap between the end of the first read and the start of the second read is +/- max-gap.

By default, when a switch-back template is identified, the primary reads are made unmapped (and the original alignment stored in the OA tag) and all secondary and supplementary alignments are discarded. This can be disabled with the --dont-unmap or -d option.

All reads from a switch-back template are also tagged with an sb tag that describes the nature of the switchback. If the template was identified base on soft-clipped sequence within a read the format is:

sb:Z:r,[read|mate],{offset},{length}

If the template is identified due to it’s tandem pair orientation then the format is:

sb:Z:t,{gap}

Inputs and Outputs

The tool takes as input a SAM or BAM file, and by default consumes from stdin. The primary output is also a SAM or BAM file, and defaults to compressed BAM on stdout. This allows the tool to be run immediately after an aligner in a pipe, e.g. bwa mem ref.fa r1.fq r2.fq | fgbio -Xmx8g --ref=ref.fa | ....

If the input is neither queryname sorted nor queryname grouped (i.e. all reads with the same name grouped together) it will be sorted into queryname order by the tool before processing.

By default the output BAM is produced in the order the reads were processed (i.e. the input ordering or queryname sorted if sorting was required). This can be overridden with the --sort-order option.

A number of text files are also produced if the --metrics option is specified. E.g. when specifying --metrics=s1.switchback the following files are produced:

1. s1.switchback.summary.txt: A table of summary metrics describing the number of reads, switchbacks, etc.
2. s1.switchback.lengths.txt: A table of the distribution of observed switchback lengths in read-based switchbacks.
3. s1.switchback.offsets.txt: A table of the distribution of observed offsets in read-based switchbacks.
4. s1.switchback.gaps.txt: A table of the distribution of gap lengths in tampl
5. s1.switchback.plots.pdf: A PDF containing plots of the distributions from 2-4.

Note: because this tool accesses the reference genome in a random manner it pre-loads the entire reference fasta into memory. As a result the tool is best run with -Xmx8g to give it sufficient memory.

Arguments

Name	Flag	Type	Description	Required?	Max # of Values	Default Value(s)
input	i	PathToBam	Input BAM file.	Optional	1	/dev/stdin
output	o	PathToBam	Output BAM file.	Optional	1	/dev/stdout
metrics	m	PathPrefix	Metrics output file.	Optional	1
sort-order	s	SamOrder	Output sort order.	Optional	1
ref	r	PathToFasta	Reference genome fasta file.	Required	1
max-offset	O	Int	Maximum offset between end the two segments of the read on the reference. Set to 0 to disable read-based checks.	Optional	1	35
max-gap	g	Int	Maximum gap between R1 and R2 of tandem reads to call a template a switchback. Set to 0 to disable tandem-based checks.	Optional	1	500
min-length	l	Int	Minimum match length of the switched back segment.	Optional	1	6
max-error-rate	e	Double	Maximum mismatch error rate of switchback match to genome.	Optional	1	0.1
dont-unmap	d	Boolean	IF true, do NOT unmap reads from switchback templates.	Optional	1	false