Has anyone tried Pico Methyl-Seq™ Library Prep Kit from Zymo?

[mbk0asis]

Hello, seqers!

Has anyone tried Pico Methyl-Seq™ Library Prep Kit from Zymo?

Since I usually handle embryos, it seems to fit our needs.
However, I'm suspicious on their protocol.
For Illumina NGS, adapters at both ends must be different, but they seemed same in the illustration.
I wonder if the figure is correct or the zymo was too lazy to color the adapters separately.

Check out the protocol scheme from zymo...


Thank you!

[nucacidhunter]

In the workflow illustration in the website and product manual they have missed P5 and P7 overlaid on the amplification primers. The correct workflow is illustrated in this poster:

http://www.zymoresearch.com/downloads/dl/file/id/634/d5455o.pdf

[mbk0asis]

Thank you. It was informative.

Still, I think two types of primers are needed for random priming since P5 and P7 cannot anneal to the same sequences unless only the 13 bases of complementary region in TruSeq adapters are used.

I hope Zymo provide more detailed information about the kit. They don't give any sequence information about the adapters.
I think I will send an email to Zymo tech support.

Thank you again, nucacidhunter.

[nucacidhunter]

Let’s speculate on reactions that takes place during library prep with this kit:

1- DNA bisulfite converted and sheared
2- First strand is synthesised by using primes with known 5’ overhang sequences and random 3’
3- Second strand synthesised with the same 1st strand reaction components and cycling conditions.
4- The result is dsDNA (and possibly some ssDNA) flanked by known 5’ sequence of PreAmp primer. This reaction is purified.
5- DNA is amplified with LibraryAmp primers which add some of remaining P5 and P7 sequences most likely up to flow cell binding motives. In this step P5 and P7 primers can bind randomly to each end of fragments. So some of fragments will be flanked by one of each primer for the next step. Fragments flanked by the same primer will not effectively amplify in the next step.

In step 2 the known sequences is most likely 13 nucleotides shared between P5 and P7 adapters. They could be mix of two primers one for P5 and one with P7 overhang, but it is less likely because annealing temperature during Amplification step is low (45C). Anyway in either approach some fragments will not make into final library due to presence of same adapters in both ends.

6- During amplification with index primer, remaining motives of both P5 and P7 is added producing sequence competent library. There is possibility that P5 full length was restored in previous step as well.