How to know that your RNA-seq is stranded or not?
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8.6 years ago
M K ▴ 590

Is there any way to detect if your rna-seq data is unstranded or stranded

RNA-Seq • 29k views
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5.6 years ago
Wayne ★ 1.5k

In case anybody currently looking comes across this post...

The easy-to-use Salmon will check for you as described here. You can see what the resulting abbreviations correspond to with a nice illustration here.

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+1 on a taking the time to post a more modern solution. I wonder if there is a way to in biostar to highlight answers like this.

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Upvoting it and/or selecting it as an accepted answer is the way to go. Commenting as you did is also helpful. Bioinformatics changes more rapidly hence we have to more proactive in marking up the most recent correct answer.

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There are some great answers posted already, but just in case you want to learn more about strandness, you can also check this previous post: Read pair orientation : Illumina TruSeq Stranded mRNA library

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Chris Fields ★ 2.2k

A few RNA-Seq QC tools will detect whether a run is strand-specific. For example, the infer_experiment.py script in the following claims to do this (never used this myself, so can't vouch for it):

http://rseqc.sourceforge.net/

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even this seems to require a BAM file to operate, so at that point one could look at the file

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Yep. Only other way I can think of is to check whether there is a strand-specific adaptor used, but this normally gets stripped off the sequence prior to the user getting their hands on it (at least our center does).

Actually, I don't recall whether the TruSeq strand-specific adaptor is the same sequence as their other non-strand-specific counterparts, but then again I've never had to worry about checking for this. Seq centers we've worked with are normally pretty explicit in telling us what protocols and adaptors they use.

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5.6 years ago

Hi

This image could help.

In stranded example reads are clearly stratified between the two strands

Of course, you need to perform the alignments, get the BAM file and visualize it in any of the software available (SeqMonk, RNAseqViewer, IGB, etc)

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8.6 years ago
Irsan ★ 7.6k
if you don't know what sample prep protocol was used you have to map your reads to a reference genome and look at the sam flags in the bam file. If it is stranded, flags 83, 99, 147 and 163 have the same abundance but in stranded, 2 of these 4 will disappear when you look at either sense or antisense genes only.
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It might be easier to map to the transcriptome than the genome. Then you know you are mapping to the sense side.

Remember that certain protocols map the first read to the sense strand and the second read to the antisense. Others do it the reverse (the first read is antisense).

Do you know the protocol that was used? You should be able to tell from that whether it is stranded. Joshua Levin has a paper from a couple years ago that compared a bunch of stranded protocols.

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I'm trying to figure out if my data is stranded or not. Salmon shows that it is, tags in bam file are 99 and 147 but when I plot it in Genome Browser there is almost no difference in expression between the strands. How could that be?

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how to look at the sam flags from BAN files?

Thanks,

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8.6 years ago
Josh Herr 5.7k

If you have a reference you could map to it to find out. There might be another way, but nothing else comes to mind.