Hi,
It is explained in several places such as here
https://bioinformatics.cvr.ac.uk/ill...mer-sequences/
that the adapters ligated to the DNA library in many library prep workflows contain a T overhang, which is complementary to an A overhang, that one needs to install on the library to be sequenced, resulting, to my understanding, in such fragments:
5'-Adapter-T-Library-A-Adapter-3'
Now, the website above mentions that
the Read1 primer is ACACTCTTTCCCTACACGACGCTCTTCCGATCT, which I think corresponds to the adapter + the additional T, and
the Read2 primer is GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, which, again, I believe is adapter + the T.
So far so good, weren't it for the fact that I have recently made my own libraries for a myseq run without adapter ligation, where I simply added P5/7 and adapter in a single PCR, and for which I used these primers:
P5: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-binding-region-3'
P7: 5’-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-binding-region-3'
I got these primers from a colleague, and as you may see, while the P5 primer contains the final T, the P7 primer does not. That means, that my PCR-made libraries looked like this:
5'-Adapter-T-Library-Adapter-3'
Confusingly, the sequencing run went just fine (and so have runs where my colleagues used these primers) and when I look into the sequenced fragments, indeed there is no extra A between the library and the 3' adapter. I checked some old sequencing data where I did follow an adapter ligation protocol, looked at forward reads that read into the adapter part, and there I see the extra A after my library.
So clearly, I must be missing something here, because if the Read2 primer indeed contains a T on the 3' end, then the read on my PCR libraries really should fail, because that final T on the primer has nothing to bind to in these constructs, will flap around, and the polymerase won't extend the primer.
I know that there are other library prep methods out there that don't rely on A/T ligation, so I wonder if anyone knows if they typically introduce that T/A anyway, or if the resulting adapters are there without the T/A? Could it be that the Read2 primer doesn't actually contain the final T and ignores base 1 if it turns out to be all As?
Thanks,
Max
It is explained in several places such as here
https://bioinformatics.cvr.ac.uk/ill...mer-sequences/
that the adapters ligated to the DNA library in many library prep workflows contain a T overhang, which is complementary to an A overhang, that one needs to install on the library to be sequenced, resulting, to my understanding, in such fragments:
5'-Adapter-T-Library-A-Adapter-3'
Now, the website above mentions that
the Read1 primer is ACACTCTTTCCCTACACGACGCTCTTCCGATCT, which I think corresponds to the adapter + the additional T, and
the Read2 primer is GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, which, again, I believe is adapter + the T.
So far so good, weren't it for the fact that I have recently made my own libraries for a myseq run without adapter ligation, where I simply added P5/7 and adapter in a single PCR, and for which I used these primers:
P5: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-binding-region-3'
P7: 5’-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-binding-region-3'
I got these primers from a colleague, and as you may see, while the P5 primer contains the final T, the P7 primer does not. That means, that my PCR-made libraries looked like this:
5'-Adapter-T-Library-Adapter-3'
Confusingly, the sequencing run went just fine (and so have runs where my colleagues used these primers) and when I look into the sequenced fragments, indeed there is no extra A between the library and the 3' adapter. I checked some old sequencing data where I did follow an adapter ligation protocol, looked at forward reads that read into the adapter part, and there I see the extra A after my library.
So clearly, I must be missing something here, because if the Read2 primer indeed contains a T on the 3' end, then the read on my PCR libraries really should fail, because that final T on the primer has nothing to bind to in these constructs, will flap around, and the polymerase won't extend the primer.
I know that there are other library prep methods out there that don't rely on A/T ligation, so I wonder if anyone knows if they typically introduce that T/A anyway, or if the resulting adapters are there without the T/A? Could it be that the Read2 primer doesn't actually contain the final T and ignores base 1 if it turns out to be all As?
Thanks,
Max