Solution To Find Out Read Orientation As Well As Strand To Which Reads Align From Sam File

Question

[k2bhide]

Hello all, I have sam files from strand specific RNASeq (generated from SOLiD platform). I would like to separate reads by forward and reverse orientation as well as by the strand to which they align. Is it possible to get this information from sam file (bitwise flags) or cigar strings etc. If so, which flags I should seach for and parse from the sam file.

Regards Ketaki

Answer 1

[Ashutosh Pandey]

The information about the forward and reverse orientation can be deduced using the bitwise flags. See this: http://picard.sourceforge.net/explain-flags.html.

Regarding strand, do you have any special tags in your BAM file that were generated by SOLiD aligner ?

[k2bhide]

By special tags do you mean bitwise flags or cigar strings?

[Ashutosh Pandey]

Tags like NM, NH. Also, which aligner you used ?

[k2bhide]

I have to check with people at core outside to know which aligner they use. I will get back to you once I have the information.

Answer 2

[Biomonika (Noolean)]

Do you have single end reads or paired end reads? What you expect when having strand-specific data for paired-end reads is that first read of the pair will align to either + or - strand for certain exon - please check my answer here: A: Should RNA-seq reads map to one strand of cDNA reference?

One of the solutions is to convert sam to bam and then filter based on flags explained here: http://picard.sourceforge.net/explain-flags.html

samtools view -f flag aln.bam (include reads with certain flag) samtools view -F flag aln.bam (exclude reads with certain flag)

Please specify what reads you have and what is your reference.

[k2bhide]

I have paired end strand specific RNASeq data from Yeast