What is Bowtie2 --norc parameter?

Question

[lvl2111]

Hello,

I am running an alignment of single-end reads, which are trimmed 20bp sequences that need to be aligned to my indexes of 20bp gRNAs.

I am trying to understand which bowtie2 parameters would be best for me, and came across the --norc parameter. I read the manual, but am still having trouble understanding whether I should be using this parameter or not. Can someone please describe its purpose in more understandable terms? And whether it is suggested to use it?

From the manual:

If --nofw is specified, bowtie will not attempt to align against the forward reference strand. If --norc is specified, bowtie will not attempt to align against the reverse-complement reference strand. For paired-end reads using --fr or --rf modes, --nofw and --norc apply to the forward and reverse-complement pair orientations. I.e. specifying --nofw and --fr will only find reads in the R/F orientation where mate 2 occurs upstream of mate 1 with respect to the forward reference strand.

Thank you so much.

Answer 1

[ATpoint]

My recommendation for mapping sgRNA/shRNA against a barcode library obtaining only perfect ungapped matches is:

bowtie2 -x ref.fa --end-to-end --very-sensitive --rdg 10000,10000 --rfg 10000,10000 --mp 10000,10000

That does sensitive end-to-end mapping putting huge penalties on any gap or mismatch. You can then use for example featureCounts for a count matrix using only reads with MAPQ 42 which is the highest bowtie2 score, hence using only these perfect alignments.

[lvl2111]

Thank you very much!