Targeted amplicon bisulfite sequencing library preparation

[kk123]

Hello,

I am a PhD student, looking a human methylation patterns. I am planning to design a targeted amplicon bisulfite sequencing, using a modified 16S protocol https://support.illumina.com/documen...15044223-b.pdf

All together, we would like to sequence 8250 amplicons (50 people, 5 brain regions each, 33 amplicons per brain region). We will do multiplex sequencing, so we need 250 combinations of indexes (50 people and 5 brain regions).
According to our regional Illumina commercial service each of the 33 amplicons per region per person should be multiplied separately with overhang primers, and then all the 33 amplicons should be equimolarly merged into one sample and multiplied by one pair of Nextera forward and reverse indexes.

Now my main question would be will the second round of PCR work? Will there be sufficient amplification concentration at the end? Can you successfully amplify 33 different amplicons simultaneously in a single well? They will be approximately similar in length (around 300bp) but still. Any input is greatly appreciated.

The plan for library preparation is as follows:
1. DNA isolation
2. Bisulfite conversion
3. PCR round one using primers with 5’ overhang (8250 reactions)
4. Gel Electrophoresis / Agilent
5. Cleaning with magnetic beads
6. Measurement of DNA concentration (fluorescence)
7. Equimolar pooling of all 33 amplicons per per person for all five brain regions
8. PCR round two to add forward and reverse indexes to pools of amplicon - 250 samples, combined two Nextera XT kits (set A and D)
9. Gel Electrophoresis / Agilent
10. Cleaning with magnetic beads
11. Measurement of DNA concentration (fluorescence)
12. Equimolar combination of all 250 samples to generate a single library

[nucacidhunter]

Yes it works and your plan looks fine. Second round of PCR adds the indexes and remaining adapter sequences and is primed from the overhang which is common among amplicons.

[kk123]

Thank you for your help!

[MMG9]

How did the sequencing go? Did you have success? I am doing something similar, but had a hard time getting successful amplification.

[kk123]

This probably won't be relevant to you anymore but the run went great. If you have any specific questions I'd be happy to help.

I designed primers using Methyl Primer Express and I checked the specificity with BiSearch (so that I would only get one specific product) and I ran them through IDT oligo analyzer tool to check for primer dimers and such.

For PCR amplification I used KAPA uracil+ polymerase. For determining the best annealing temperature I used thermal gradient PCR with 12 different temperatures. Majority of primers worked well enough to produce amplicons.