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poorphd 10-18-2011 07:50 AM

how to define a forward or reverse read file
i often meet the concept of "forward" or "reverse".
but not exact definition provided

does the "forward" means the reads who have the same direction with PCR primer 1 ?
does the "reverse". means the reads who have the same direction with PCR primer 2 ?

thank u very much
shan gao

Soleil 11-21-2011 06:15 AM

In relation to your sequence:
Forward: 5' to 3'
Reverse: 3' to 5'

poorphd 11-21-2011 06:50 AM

no, not that simple
In classical biology, we define "Forward" is the direction from 5' to 3' and "Reverse". 3' to 5'. but in NGS sequencing, all the sequences direction is from 5' to 3' , so F means sense strand, and R means anti sense.but in classical biology, we define sense strand is mRNA direction. those concepts are a little confusing.

and also in the Trinity algorithm, i think sense or antisense is just oppossite to each other. if you define one srtand is sense, the other one is antisense. trinity donot care which one is which one. i think you design this parameter --SS_lib_type, just to make the output sequences following
the sense direction which defined by users.

swbarnes2 11-22-2011 01:34 PM

I would agree with Soleil...the way people use forward and reverse in NGS context, the forward read is the one that is in the same direction as your reference, and the reverse is the one that is in the opposite direction. Of course, what might be forward for a genone might be reverse when talking about a transcript.

If you were doing something other than an ordinary genomic prep, like you were doing PCR, with the adaptors incorporated into the PCR primers, and putting that product onto the flow cell, then there would be a solid correlation between read 1 and the direction of the read itsself, with respect to the reference. But with ordinary genomic-type preps, the DNA gets sheared, and adaptors are ligated, and they don't know which way the DNA was oriented with respect to the telomere, or whether your reference puts the telomere at the start or the end of your refeence. So reads go in all directions. So really, all that matters is that for each cluster, read 1 runs one way, and read 2 runs the other way; towards each other in paired end, away from each other in mate-pair.

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