Hi all,
My boss and I are having a debate on a protocol that I need a little clarity on, and hopefully someone can give the best answer.
The problem: we are interested in using RNA-seq data and looking at strand specific expression data. But, all of the sequence data that we have is from paired-end RNA sequence.
My boss thinks that this isn't a problem and we can do one of two things. either a) only use the forward sequence reads and look at the data from that, or b) simply take the reverse reads, and use the reverse-compliment of them and treat them as forward reads.
My answer is that he is wrong in both cases. By doing paired-end sequencing, you inherently loose the strand information and cannot do any strand specific information. Simply reversing the two reads will not work, and you do not know which strand the forward (or reverse read came from). It is not always the case that the forward read is the plus strand and the reverse read is the minus strand, and these can be swapped. Essentially, we do not know where (strand and orientation) that the read came from.
Can someone help confirm that I am correct .
My boss and I are having a debate on a protocol that I need a little clarity on, and hopefully someone can give the best answer.
The problem: we are interested in using RNA-seq data and looking at strand specific expression data. But, all of the sequence data that we have is from paired-end RNA sequence.
My boss thinks that this isn't a problem and we can do one of two things. either a) only use the forward sequence reads and look at the data from that, or b) simply take the reverse reads, and use the reverse-compliment of them and treat them as forward reads.
My answer is that he is wrong in both cases. By doing paired-end sequencing, you inherently loose the strand information and cannot do any strand specific information. Simply reversing the two reads will not work, and you do not know which strand the forward (or reverse read came from). It is not always the case that the forward read is the plus strand and the reverse read is the minus strand, and these can be swapped. Essentially, we do not know where (strand and orientation) that the read came from.
Can someone help confirm that I am correct .
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