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Old 05-17-2017, 03:36 AM   #21
jchoo
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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?
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Old 10-03-2017, 02:43 PM   #22
Buckethead84
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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?

Last edited by Buckethead84; 11-24-2017 at 10:35 PM.
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Old 12-18-2017, 01:47 AM   #23
korostin
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Originally Posted by epistatic View Post
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?
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Old 12-18-2017, 01:53 AM   #24
korostin
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Quote:
Originally Posted by Buckethead84 View Post
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.
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Old 12-31-2017, 02:31 AM   #25
HiroMishima
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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
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Old 01-09-2018, 01:27 AM   #26
korostin
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Originally Posted by HiroMishima View Post
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)
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Old 01-09-2018, 09:04 AM   #27
omgbioinfo
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Illumina now sells an AmpliSeq kit compatible with their systems through a partnership with ThermoFisher: https://www.illumina.com/products/by.../ampliseq.html
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Old 01-09-2018, 09:10 AM   #28
korostin
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Quote:
Originally Posted by omgbioinfo View Post
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
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Old 01-09-2018, 04:22 PM   #29
HiroMishima
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Thank you for your question.

Quote:
Originally Posted by korostin View Post
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.
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Old 01-12-2018, 11:14 AM   #30
korostin
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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?

Last edited by korostin; 01-12-2018 at 11:17 AM.
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Old 01-14-2018, 06:17 PM   #31
HiroMishima
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Quote:
Originally Posted by korostin View Post
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?
In our protocol, we performed AmpliSeq multiplex PCR using the following materials:
1) original AmpliSeq primers: containing target-specific sequences with uracil nucleobases but not containing universal sequences
2) KAPA2G FAST Multiplex Kits: uracil-tolerant polymerase and regular dNTPs (A, C, G and T)
3) genomic DNA

The uracil DNA glycosylase and endonuclease IV treatment for the amplicons followed by the AMPure XP cleaning obtained blunt-ended uracil-less fragments being ready for Illumina library construction.

I hope that answers your question.

Hiro.
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