[marlon4711]

Hi Buzz0r,
did you use the Ion AmpliSeq HiFi Taq mix for the PCR of the panel or another enzyme. I was trying to circumvent buying the Ampliseq mix and I tried to amplify the panel with kappa mix and I didn't observe any products even after more cycles.
Thanks and all the best,
Marlon

[dna-expert]

The problem of multiplex PCR with Ampliseq primer set is U-containing primers. Since almost polymerases can not go through dU, it`s not so easy to substitute ampliseq library prep kit. NEB Next, Q5-polymerase and other NEB enzymes don`t work with U-containing primers.
Dont ask me what polymerase is ok. I still don`t know)

[epistatic]

https://www.thermofisher.com/order/catalog/product/F555S

[dna-expert]

epistatic, thank you!
found even more suitable one (multiplex compatible)

https://www.thermofisher.com/order/catalog/product/F562S

[GRClark]

Hi Guys,

Just wondering if anyone has used either of those Phusion polymerases with Amp on MiSeq workflows yet? If so, any comments you can share on their suitability?

Many thanks

Graeme

[jchoo]

hi,
Have anyone try analysis the error rate of per base in amplicons? We found lots of sites with error rate larger than 0.01, any idea why this happen?

[Buckethead84]

Hi,

Has anyone gotten this protocol to work? I have a protocol that uses Phusion U MM (thanks to epistatic) that appears to work, but I haven't tried it without the end repair reaction yet. Do you think the end repair is necessary?

[korostin]

https://www.thermofisher.com/order/c.../product/F555S

Ok, now we know how to amplify PCR-products. I think primer digestion process consist of two reactions. First, uracil-glycosylase https://www.thermofisher.com/order/c...product/EN0361 hydrolyze U from 5'-strand and we have nick. I am still wondering what would be second reaction where strand opposite to nick digested. Do you have any ideas?

[korostin]

Hi,

Has anyone gotten this protocol to work? I have a protocol that uses Phusion U MM (thanks to epistatic) that appears to work, but I haven't tried it without the end repair reaction yet. Do you think the end repair is necessary?

I think end-repair would be useful, especially PNK treatment – primers usually are not phosphorilated.

[HiroMishima]

Hi all,

In the following paper, we described a technique to prepare Illumina libraries using AmpliSeq amplicons:
J Hum Genet https://www.nature.com/articles/s10038-017-0392-9
PubMed https://www.ncbi.nlm.nih.gov/pubmed/29279608

[korostin]

Hi all,

In the following paper, we described a technique to prepare Illumina libraries using AmpliSeq amplicons:
J Hum Genet https://www.nature.com/articles/s10038-017-0392-9
PubMed https://www.ncbi.nlm.nih.gov/pubmed/29279608

Thanks for links to your article!

1. As I understand from your article you decided to avoid cuttting ampliseq primers. Why?
2. Why you used uracil depletion technique instead of U-passing with Phusion U from Thermo with average dNTP's? In total U-containing part of library after PCR would be tiny (and would not form clusters on flowcell)

[omgbioinfo]

Illumina now sells an AmpliSeq kit compatible with their systems through a partnership with ThermoFisher: https://www.illumina.com/products/by.../ampliseq.html

[korostin]

Illumina now sells an AmpliSeq kit compatible with their systems through a partnership with ThermoFisher: https://www.illumina.com/products/by.../ampliseq.html

We aren't looking for easy ways)) Price per 1 library more 100$ – it's too expensive

[HiroMishima]

Thank you for your question.

Thanks for links to your article!

1. As I understand from your article you decided to avoid cuttting ampliseq primers. Why?
2. Why you used uracil depletion technique instead of U-passing with Phusion U from Thermo with average dNTP's? In total U-containing part of library after PCR would be tiny (and would not form clusters on flowcell)

A1) We did AmpliSeq primer cutting. For amplicons, we used uracil DNA glycosylase and endonuclease IV. This step obtained amplicons without primers outside innermost uracil.

A2) Because we wanted to use regular polymerases already used in related protocols. This may be important for researchers using other protocol hacks . I agee with you. Uracil-tolerant polymerase may work.

Hiro.

[korostin]

Hiro,

Maybe I don't understand main Ampliseq feature correctly? Because for my mind, super-multiplex provided by step-out PCR based on universal identical 5'-tail on all primers. So, primer cleavage means 5'-tail cutting. Also, universal and specific parts of every primer separated by uracil. After PCR all products would be like:

5' universal-tail-U-NNNNNNNNNNNN......................... 3'
3' .......................NNNNNNNNNNNN-U-5'universal-tail 5'

[dots mean nothing – only for visualisation]
Because classic DNA polymerases can't read uracil and stop synthesis.
If so, uracil DNA glycosylase and endonuclease IV treatment would produce blind ends. If PCR products have no 5' overhangs, uracil DNA glycosylase and endonuclease IV would create nicks only.

Could you explain, please?