Calling SNPs from bacterial RNA-seq

[ThePresident]

Hello everybody,

It's been a while and I'm back with a question.

I am dealing with bacterial RNA-seq data (Illumina, single read, 2 replicates conditions A and B). For convenience, I use Artemis to rapidly visualize RNA-seq coverage. Now, with Artemis, there's an option for SNPs marking, and I observed that some genes are highly variant, i.e. there is a strong concentration of SNPs in one or few regions. When I simply zoom in (until I see my reads as sequences), I can see that some of my reads have a point-mutation here and there but never all of the reads on the same place.

1. 1 I wonder if these observations of SNPs are genuine or it's only a mere artifact?
2. 2 Is there a simple way of calling SNPs genome wide, or even on a specific region (ex. on my gene of interest?)

Thank you in advance,

TP

[swbarnes2]

Hello everybody,

It's been a while and I'm back with a question.

I am dealing with bacterial RNA-seq data (Illumina, single read, 2 replicates conditions A and B). For convenience, I use Artemis to rapidly visualize RNA-seq coverage. Now, with Artemis, there's an option for SNPs marking, and I observed that some genes are highly variant, i.e. there is a strong concentration of SNPs in one or few regions. When I simply zoom in (until I see my reads as sequences), I can see that some of my reads have a point-mutation here and there but never all of the reads on the same place.

1. 1 I wonder if these observations of SNPs are genuine or it's only a mere artifact?
2. 2 Is there a simple way of calling SNPs genome wide, or even on a specific region (ex. on my gene of interest?)

Thank you in advance,

TP

In a real SNP, you should see a bunch of reads, starting at different places, running both directions (if your prep isn't strand specific), supporting the alternate call. If you don't see that, but just sporadic differences here and there, that's just Illumina error.

samtools mpileup will call SNPs. GATK will too, but it might be a bit more involved.

[ThePresident]

Thank you,

I'll try mpileup!