Some confused from samtools mpileup.

[fabrice]

When I run samtools with follow parameters:

samtools mpileup -C50 -Q25 -q1 -l %s -f %s %s

I got a output as follow:

21 34124586 G 38 ,,,,AAA,AAAAa..,,,aaa.AaA,,A,,aaaaaaaA

From samtools manual:

"At this column, a dot stands for a match to the reference base on the forward strand, a comma for a match on the reverse strand, a ’>’ or ’<’ for a reference skip, ‘ACGTN’ for a mismatch on the forward strand and ‘acgtn’ for a mismatch on the reverse strand."

How can I see 'A' at the forward strand, then see 'A' at the reverse strand?

From the result, at position chr21:34124586, it seems it is heterozygous snp A/G. If the gene is expressed on the forward strand. So we should only see A or G on the forward strand. Why it can mapped to the reverse strand. Because only the forward strand DNA has been transcript to mRNA. The reverse strand does not.

I am a bit of confused here. Could any one give me some hits?

Thank you very much in advance.

[swbarnes2]

When I run samtools with follow parameters:

samtools mpileup -C50 -Q25 -q1 -l %s -f %s %s

I got a output as follow:

21 34124586 G 38 ,,,,AAA,AAAAa..,,,aaa.AaA,,A,,aaaaaaaA

From samtools manual:

"At this column, a dot stands for a match to the reference base on the forward strand, a comma for a match on the reverse strand, a ’>’ or ’<’ for a reference skip, ‘ACGTN’ for a mismatch on the forward strand and ‘acgtn’ for a mismatch on the reverse strand."

How can I see 'A' at the forward strand, then see 'A' at the reverse strand?

From the result, at position chr21:34124586, it seems it is heterozygous snp A/G. If the gene is expressed on the forward strand. So we should only see A or G on the forward strand. Why it can mapped to the reverse strand. Because only the forward strand DNA has been transcript to mRNA. The reverse strand does not.

I am a bit of confused here. Could any one give me some hits?

Thank you very much in advance.

It doesn't mean the revcomp of the reference strand. 'A' means that you have reads that align in the forward direction that have an A there. 'a' means that you have reads that align in the reverse direction that have an A there. If the only reads that had an A at that locus were all in the same direction, that's a sign that it's not a real SNP, but some kind of misalignment.

Unless you specifically did a strand-specific library prep, and you probably didn't, you should expect to see reads in both directions, even though your mRNA is only in the one direction.

So your SNP looks fine, and real, and normal.

[fabrice]

swbarnes2,

Thank you for your clarify. Yes. I did not do a strand-specific library prepare. But I still have bit of problem to understand why I should expect to see reads in both directions.
The mRNA is only in the one direction, when sequence we should also see one direction.