Specific-strand Sequencing vs. Anti-sense Sequencing

[SeqGirl7]

I need some explanation on strand specific sequencing. If I'm not mistaken, mRNA strands obtained from sample will be added with adaptors at both 3' and 5' end to conserve the RNA sequence. From here, ligated RNA will under reverse transcription generating cDNA followed by PCR amplification.

However, I was informed by sequencing company that this strand-specific sequencing is also known as anti-sense sequencing. I am confuse when they told me this.

The strand that will be sequence is cDNA generated from mRNA right? or both amplified strand (cDNA and reverse strand of cDNA). I think I'm confuse with the concept here.

Appreciate any explanation on this.

[blanco]

The standard procedures for library preparation protocols work by first generating cDNA from the mRNA which is then followed by adaptor ligation and PCR amplification. By this method you lose information about from which strand the mRNA is transcribed from and should get roughly equal reads from both strands.

By preparing strand specific libraries, for example by using dUTP in which case only the first strand from cDNA synthesis in PCR amplified, you conserve information from which strand the mRNA is transcribed and the read from both strands are not equal. So if you have reads from the same place but say 80% is from the plus strand and 20% is from the minus strand you can tell you have 80% sense transcription and 20% antisense transcription. So from that perspective, one can say that strand specific sequencing is antisense sequencing but it is more than that. With strand specific sequencing you can tell from which strand your reads come from regardless of whether there is any antisense transcription or not.

This is only my first post here so maybe some more experienced user can confirm and/or expand on my explanation.

[pmiguel]

That sounds right to me, Blanco. I guess one could add there are multiple methods of creating strand specific libraries. Also, I have not heard "strand specific" libraries referred to as "anti-sense" sequencing. Nor do I think that would be an apt naming.

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Phillip