What sequence do I get when using paired-end stranded protocol
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10 months ago
Assa Yeroslaviz ★ 1.5k

I know this is a very basic question, but I tend to mix them always. I was wondering what reads Do I get at the end, when I run a paired-end directional (or stranded) library preparation protocol.

I know from here, that in a single-end directional protocol the reads I see in the fastq files are the original transcripts, which are in the genome. (as opposed to the un-directional protocol, where one can't say from which strand the reads is coming from)

But what is happening when I'm sequencing a paired-end directional protocol?

Which of the two reads from each pair is which?

Can I make these statements?

  1. When doing a single-end (SE) directional library, The reads I get in the fastq files are the original mRNA sequences.
  2. When doing SE un-directional sequencing there is no way of telling if the reads I see in the fastq files are from the sense or anti-sense strand

  3. When I have a paired-end sample done with directional library prep., R2 of my sample is the original mRNA molecule, while R1 is the anti-sense

thanks Assa

RNA-Seq directional stranded paired-end sequencing • 439 views
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Entering edit mode
10 months ago
caggtaagtat ★ 1.4k

To statement 1: No, there are 2 major types of SE stranded (directional) libraries. Forward stranded and reverse stranded. If your library only consists of reverse stranded RNA fragments, you get the original RNA sequence in the reads through the sequencing, since it is sequencing by synthesis. Also you don't always have only mRNAs in the data, only if you either enrich them before library preparation.

Statement 2: With stranded SE libraries you also don't know wether the read comes from the genomic sense or antisense strand, but you at least know if the read is in sense or antisense to the original RNA fragment. So if you meant that with unstranded SE libraries, you don't know whether the read is in sense or antisense orientation to the sequenece of the RNA fragment, than yes.

Statement 3: Again, there are more than 1 type of paired-end stranded (directionaly) libraries availible, "Invard", "Outward" or "matching". They are nicely explained in the documentation of salmon. Basically one read of a pair covers the sequence of one end of a specific RNA fragment and the other read of the pair covers the sequence of the other end of that fragmen, either facing each other or not. I don't know exactly what "matching" means.


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