ssRNA-seq:Why saying ignoring the orientation of transcription leads to an overestimation of expression level for genes where the level of reverse transcription is comparable with that of forward

Question

[harrypotterandsbt]

Hello everyone ,It 's my first question in biostars. Recently I was annoyed by a question about ssRNA-seq library preparation. there are two question : 1. we know that standard RNA-seq library preparation produce double-strand cDNA.Does two strands of cDNA all be sequenced in sequencing process ? If so, then whether sequencer do the repetitive sequenceing ? I read a sentence from a paper called 《Transcriptom analysis by strand-specific sequencing of complementary DNA》it says：ignoring the orientation of transcription leads to an overestimation of expression level for genes where the level of reverse transcription is comparable with that of forward。Whether it means standard RNA-seq also count antisense transcripts as sense transcripts(if antisense transcripts exiested) ,and that leads to the overestimation of expression level of genes.

2.we know that strand-specific RNA-seq library preparation will get ride of second strand of cDNA to retain the orginal infromation but then cDNA first strand will PCR and synthesize second strand but we know that in sequencing precess ,only the first strand will be sequenced . I wonder whether the special 5' and 3' adapter will not only be used to retain directional information of cDNA first strand ,but also play a role in P5 P7 adapters in sequencing in illumina platform? I refer the A: RNA seq stranded
I hope someone who knows the answer can help me .Thanks advance!

Answer 1

[Kevin Blighe]

That manuscript is quite old. Here it is for others: Transcriptome analysis by strand-specific sequencing of complementary DNA.

In un-stranded RNA-seq library preparations, mRNA deriving from the sense and anti-sense strands are sequenced indiscriminately. That is, there is no distinction made between them. During analysis stage, the reads deriving from these are then aligned to the reference genome (or pseudo-aligned to the reference transcriptome).

In stranded RNA-seq library preparations, extra steps are used such that we can correctly distinguish sense mRNA from anti-sense mRNA.

If you think about it, with un-stranded library preparations, over-estimation of count abundance is a real possibility because, for many regions of the genome / transcritpome where transcription on both sense and antisense overlaps, we will not know if the aligning read from our RNA-seq run was originally derived from the sense or anti-sense strand.

Kevin

[harrypotterandsbt]

Thank you Kevin Blighe! You have solved my first question.And Do you know the second question's answer?

[Kevin Blighe]

Can you try to re-phrase your second question, perhaps?

[harrypotterandsbt]

OK, What I want to express is that in stranded RNA-seq library preparation, the left cDNA first strands are still need to PCR before sequencing. So we still will synthesize the second strands ,but only the first strands are selected to sequencing. There must be a selective mechanism. And I think it must be relative to the special adapters that are added to the first cDNA strand ?Can you give some more details information ?Thanks!

[Kevin Blighe]

Yes, there are special adapters used in the stranded process, which also involves the use of dUTP degradation.

It is important to remember that sense transcription occurs in one direction, whereas anti-sense transcription occurs in the opposite direction. This is the key information that allows for strand-specific protocols:

• In un-stranded, the adapters that are used do not retain information on the direction of transcription.
• In stranded, the special adapters, via dUTP degradation, retain information on the direction of transcription. Thus, they also can distinguish sense from anti-sense.

Each stranded protocol differs. The manufacturer's likely have the most precise information.

[harrypotterandsbt]

I get it ! Thank you! Kevin Blighe. I really appreciate your help !