How Is It Possible For Adapter Sequence To Be Present At The Beginning Of The Reads?

Question

[lwc628]

I grepped the adapter sequence to the fastq file with coloring option, and found out that there are a lot of reads that contain adapters. But most of them are on the beginning of the reads(5' end) rather than at the end (3' end).

How is it possible for adapter sequence to be present in the beginning of the reads? I thought the mechanisms of the adapters is that when the sheared mRNA pieces ligated with adapters are shorter than the sequencing length. When the sequence of interest is shorter than the sequencing cycle, machine sequences into the adapter ligated to the 3' end of the each molecule. If this true, we should only expect to see the adapter in the 3'end, correct?

Where do my mental model falls short?

Answer 1

[Mikael Huss]

I think it could be due to adapter dimers. Both ends of the fragments (5' and 3') have adapters, but the one at the 5' end is usually not visible in the read sequence because that's where the sequencing process starts. If you have an adapter dimer, however, the polymerase can attach to the first adapter, reading through the second one, if you understand what I mean.

[lwc628]

It makes sense. So are you saying the adapters in the 5' end are rare events? What if I see them a lot?!

[Mikael Huss]

They should typically be rare events. After all the adapters are designed to avoid this. Now I am really on thin ice (because I couldn't pipette my way out of a paper bag) but my impression is that if the library prep works badly, so that you don't get good enrichment of the material you want, then you will see a lot of adapter dimers.