Question: SAM Flags for illumina paired-end reads mapped on transcriptome
gravatar for Macspider
13 days ago by
Vienna - BOKU
Macspider1.5k wrote:

I am mapping illumina paired-end reads against a transcriptome with HISAT2 and I'm having a moment of confusion understanding one thing which might be easy actually:

Is it normal to have sam flags that point at the same strand for both reads? The technology output should produce one read that maps on the forward and one read that maps on the reverse strand (when mapping against a genome) but I'm uncertain of what I should observe when mapping against a transcriptome.

I guess the program should map one of the two mates and reverse-complement the other, but does the output flag (and mapping result in general) reflect the mapping strand of the reverse-complement or of the original read? is it normal to have many pairs with both mates on the same strand?

Please help me out of this "theoretical" quicksand.

ADD COMMENTlink modified 13 days ago by Devon Ryan71k • written 13 days ago by Macspider1.5k
gravatar for Devon Ryan
13 days ago by
Devon Ryan71k
Freiburg, Germany
Devon Ryan71k wrote:

It doesn't matter what you're aligning against, you expect PE mates to align with opposite orientations. The strandedness of the underlying data plays absolutely no role in this (your reads don't have strands they have orientations). Unless a very atypical library prep. was used, the flags you should commonly see in the resulting BAM file are 99, 147, 83, and 163. If those don't constitute the overwhelming majority of the flags then something likely went amiss.

ADD COMMENTlink written 13 days ago by Devon Ryan71k
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