3 prime tag counting RNA Seq

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6.1 years ago

Payal ▴ 160

Hi,

I am currently working on an RNA Seq dataset, which has some 3 ' bias (I found that out by Picard RNASeq Metrics). So I was suggested to perfotm 3 prime tag counting.

I came across this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3954844/

Can someone please explain to me how they are changing the GTF based on expression and doing the length restriction?

Thanks, Payal

RNA-Seq • 2.2k views

ADD COMMENT • link updated 5.9 years ago by Biostar 20 • written 6.1 years ago by Payal ▴ 160

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How biased is your data? some 3' bias is not that uncommon in any RNAseq dataset

ADD REPLY • link 6.1 years ago by lieven.sterck 15k

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This is how the graph looks,https://ibb.co/c7ou2H.. Is this normal?

ADD REPLY • link 6.0 years ago by Payal ▴ 160

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OK, there is indeed 3' bias but would not immediately worry about it. We are talking about mapping against transcripts, right? (de novo ones or genome derived?) How did you made the graph/plot?

as Wouter said: it this consistent over all your samples?

ADD REPLY • link 6.0 years ago by lieven.sterck 15k

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Yes its consistent among all samples..I aligned the fastq files to reference genome via Hisat2. I used Picard RNASeq Metrics to plot this graph.

I just got to know this is FAC sorted data and it seems FAC sorted samples show this kind of 3prime bias..so i think i have to figure out how to do 3 prime tag counting!!

ADD REPLY • link 6.0 years ago by Payal ▴ 160

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I wouldn't worry too much about that bias, except if there is markedly difference in the extent of the bias between samples/conditions.

ADD REPLY • link 5.7 years ago by WouterDeCoster 47k

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