Very confused about strand-specific

[msfep]

Hello,

I've been thinking a lot on how to align strand specific RNA-seq data since I have dUTP RNA-seq libraries (read 1 reverse and read 2 forward). I've produced a de novo transcriptome with Trinity and know I was using RSEM to map reads to this transcriptome. While reading this topic where someone says:

"For your protocol you should simply use --forward-prob 0 (and not use --strand-specific).  When this option is set to zero, RSEM will turn on the --nofw option for Bowtie."


...I was confused.
The bowtie manual says that:
"In paired-end mode, --nofw and --norc pertain to the fragments; i.e. specifying --nofw causes bowtie2 to explore only those paired-end configurations corresponding to fragments from the reverse-complement (Crick) strand."

What I understand from this is if you have a reference genome and you know what is the sequence from the Crick strand and from the Watson strand, you might want to know how many reads pairs map to the Crick strand and so you set this option on. In de novo assembly case, Trinity only outputs sequences which might be Watson or Crick, but we don't know which is which. But, since our read1 will only aligns to reverse strand of Watson or Crick strand, you set the --nofw flag forcing then Bowtie to align read1 to the reverse strand and reverse complementing read2 to align it too? Is this what is happening?

What is making me confused is that if run the Bowtie command from RSEM without the --nofw option I get double the alignments. In this case, Bowtie also assumes aligments of reverse complement read 1 to the forward strand?

Thanks

[msfep]

Ok, I think I already answered myself (I think I might not be able to explain it really well but I can try if someone is interested). But can someone confirm me if the --nofw behavior will be searching for aligments of read1 in the reverse strand of a reference sequence and then pairing read2 in the forward strand?

Mafalda

[Bo Li]

Hi Mafalda,

Yes, it is. You can confirm it by reading the Bowtie/Bowtie 2 manual.

Hope it helps,
Bo

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