Need Help. Paired-end Adapters for GBS

[Chimplint]

Howdy!

Need some help with a Genotyping-by-Sequencing (GBS) project. Previously, we successfully sequenced a GBS library using the barcode/common adapter setup (Elshire et al), which was not suitable for paired end reads. The adapters were then modified to follow the Illumina Y adapter formation for paired-end reads, as used with GGRS (by Qiang Chen et al). I now have a problem where I do not see the desired library, just primer and adapter dimers (at ~80 and 125 bp) and an unknown fragment curve at about 1500-3000 bp.

Troubleshooting to date:
1) Checked new adapter sequences for errors. Could not find anything that would explain my problem. Sequences are the same as Illumina's adapters for paired-end gDNA sequencing, with the addition of barcode and complementary restriction enzyme overhang in proper locations.
2)Ran digestions using new and previously used reagents/enzyme. Results: all digestions were successful; concluded that was not a contributing factor.
3) Used digestion products from above to test ligation and PCR, one set of rxns with the barcode/common adapter (successfully used in original protocol) and another set with the new barcoded Y adapter. Results: the barcode/common adapter produced the desired and expected library. The barcoded Y adapter failed, resulting in only primer and adapter dimers and the unknown fragment curve at ~1500-3000 bp.

Based on troubleshooting so far, I am confident it is NOT the starting DNA, reagents, or enzymes being used as they all work well with the barcode/common adapters.

My next experiment is to try phosphorylating the adapters, as this was done in the GGRS method and not the GBS. I have been reluctant testing this b/c restriction enzyme digestions result in 5' phosphorylated DNA fragments and it seems unnecessary to phosphorylate the adapters as well. Any thoughts?

Articles used to develop/modify protocol:
Elshire et al - PMCID: PMC3087801
Qiang Chen et al - PMCID: PMC3715491

[nucacidhunter]

If adapters’ P7 strand is not phosphorylated as you mention and you are using HotStart DNA polymerase, P7 strand will dissociate during initial polymerase activation temperature and with only one ligated adapter PCR will fail.

[SNPsaurus]

Can you tell if the Y adapter has annealed properly?

[nucacidhunter]

Success of annealing can be checked by using PicoGreen or other dsDNA specific dye, but for checking annealing PROPERLY, I do not know any assay but ensuring sequence complementarity in required region.

[nucacidhunter]

Success of annealing can be checked by using PicoGreen or other dsDNA specific dye, but for checking annealing PROPERLY, I do not know any assay but ensuring sequence complementarity in required region.

Just a second thought, maybe digesting annealed oligos with Exonucleases that use ssDNA as substrate but are inactive against dsDNA can be used for developing an assay.

[Chimplint]

Phosphorylating Adapters Did NOT Work??

Well, I phosphorylated my adapters and it did not resolve my problem...only made it worse. In the experiment, I phosphorylated both the original barcode/common adapters and the new Y adapter. I setup and ran 4 rxns using both sets of adapters with and without phosphorylation.

The only rxn that lead to the desired library was the original adapters without phosphorylation. Both phosphorylated adapter rxns resulted in a massive adapter dimer peak at ~125 bp.

Note: I'm using restriction enzyme to fragment DNA, which I have read on NEB and Promega's websites will leave 5' phosphorylated ends at cleavage sites. Does anyone know if kinase (T4 PNK is what I'm using) requires a phosphate group on both strands being ligated or just one strand? In other words, I've read that a phosphate group is needed on the vector (adapter in my case) OR insert, but not both.

Also, can't figure out why the barcode/common adapters work without phosphorylation and not the Y adapter?

Thanks again for any help/replys.

[nucacidhunter]

Note: I'm using restriction enzyme to fragment DNA, which I have read on NEB and Promega's websites will leave 5' phosphorylated ends at cleavage sites. Does anyone know if kinase (T4 PNK is what I'm using) requires a phosphate group on both strands being ligated or just one strand? In other words, I've read that a phosphate group is needed on the vector (adapter in my case) OR insert, but not both.

Also, can't figure out why the barcode/common adapters work without phosphorylation and not the Y adapter?

What you have read on those web sites is correct. Are you saying that you are using kinase for ligation? I would suggest looking at this page:https://www.neb.com/products/m0201-t...leotide-kinase
If a PCR fragment is ligated to a vector for transformation the fragment does not need 5' phosphate because the nick will be repaired in E coli. In your GBS experiment top strand (P5) does not need a 5' phosphate because its 3' end will be ligated to restriction fragments 5' end that has a phosphate. But bottom strand will need to have a 5' phosphate for ligation to restriction fragment's 3' end. If you use standard polymerase with initial incubation at 72C as Elshire protocol (not a HotStart) this is not necessary because it will be repaired by polymerase before denaturation. But if you use a hot start polymerase that nick will not be repaired and will result in dissociation of P7 and PCR failure.

More information about workflow would help trouble shooting.

[Niels Wagemaker]

Don't use phosphorylated adapters, too many primer dimers...
You can insert a quick nick translation reaction by Dna polymerase I but i only do this because of the epiGBS bisulphite reaction, you don't need to do this if you don't use hot start polymerase ( altough i get GBS libs while using hotstart taq... i think the adapter part that get melted off will reanneal in time for PCR...)

Use a different version like in : epiGBS: reference-free reduced representation bisulfite sequencing
doi:10.1038/nmeth.3763

But no methylated cytosines of course... I get perfect GBS and epiGBS libs this way... (in a 12*12 barcode setup)

[ecSeq Bioinformatics]

DNA Methylation Data Analysis Workshop
How to use bisulfite-treated sequencing to study DNA methylation

When?
22. - 25. November 2016

Where?
Leipzig, Germany

Link?
http://www.ecseq.com/workshops/works...-Data-Analysis