samtools mpileup forward vs. reverse strand?

[says_anova]

Hi all,

Apologies if this has been addressed in an earlier post, I'm definitely a n00b. Just need a sanity check:

Working with RNASeq data, the mpileup output of reads assigns '.' to a reference match on the "forward strand" and a ',' for a reference match on the "reverse strand."

In this case, the "forward" and "reverse" terminology doesn't reflect the double strandedness of DNA, but the cDNA strands being sequenced (as complements to the mRNA from the sample), where the "forward" and "reverse" tells you something about primer alignment and sequencing.

Do I have that right??

Thanks in advance!

[says_anova]

Can anyone out there confirm or deny?

[swbarnes2]

Honestly, I don't get your question. And bumping it won't fix that, or make it more attractive to people who might answer it.

Unless you do a strand-specific RNA prep, you should have half forward reads, half reverse reads, at a given locus, even though your RNA is obviously stranded.

[says_anova]

swbarnes2 - Sorry if the question wasn't clear. i think I'm confused on some basic mol. bio.

When you say half forward and half reverse reads, what are they forward and reverse with respect to?

Thanks in advance.

[swbarnes2]

swbarnes2 - Sorry if the question wasn't clear. i think I'm confused on some basic mol. bio.

When you say half forward and half reverse reads, what are they forward and reverse with respect to?

Thanks in advance.

In respect to your reference fasta. If your reference fasta says

AGTAGTATAGGTA

and the fastq has a read like:

AGTAGTATAGGTA

That's forward. If the fastq had this as the sequence:

TACCTATACTACT

That aligns in the reverse direction.