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Old 05-17-2017, 02:36 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, 01:43 PM   #22
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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 09:35 PM.
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Old 12-18-2017, 12:47 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, 12:53 AM   #24
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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, 01:31 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, 12:27 AM   #26
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Quote:
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, 08:04 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, 08:10 AM   #28
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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, 03:22 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, 10:14 AM   #30
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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 10:17 AM.
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Old 01-14-2018, 05:17 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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Old 05-13-2018, 04:12 PM   #32
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Quote:
Originally Posted by korostin View Post
We aren't looking for easy ways)) Price per 1 library more 100$ it's too expensive
Yeah I've been looking at alternatives too but it's hard to beat the sequence quality and workflow simplicity.

I'll try out a DIY ampliseq approach using NEB enzymes with some modifications:
1. OneTaq Hot Start Master Mix with GC buffer should be sufficient for the multiplex PCR reaction with uracil-containing ampliseq primers. I would scale the reaction down to 20 ul and run a 4/8/16 minute anneal extend. NEB recommends a 68C extension however such a long annealing should provide enough extension activity. Also annoying that Thermo calls their PCR enzyme "AmpliSeq Hifi" when it probably isn't even a high fidelity enzyme.
2. There was a post suggesting using USER and Exonuclease I for the FuPa digest however it was never tested and I'm not sure what the difference is between USER and UDG. I would simply add 10 units of UDG and Endonuclease IV directly to each PCR reaction and incubate at 37C for 1 hour. Adding 3' dA tailing would be convenient for Illumina adapter ligation
3. Blunt/TA Ligase master mix, scaled up to ~ 50 ul though it might be possible to find a 5x mix to keep the ratios as similar to Thermo's as possible. This would work with Ion Xpress adapters or off-the-shelf Illumina barcoded adapters if dA-tailing was performed.
4. Ampure wash, same as Thermo's recommendation but scaled up.
5. Library amp with Q5 Hot start HiFi 2x master mix and P1/X primers or P5/P7 primers.
5. Final size selection and ampure wash as per Thermo's guidelines.

The total cost of the NEB enzymes came out to around $8 per sample. I would order custom P1/X primers from IDT as well as off-the-shelf P5/P7 primers off IDT. Most expensive components would be the AmpliSeq primers followed by the sequencing adapters. The total cost per sample would still be far less than the $100/sample Thermo and Illumina are charging for without the AmpliSeq primers or barcoded sequencing adapters.

Let me know if I'm missing anything.

Ivan
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Old 05-22-2018, 08:56 AM   #33
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Quote:
Originally Posted by idedios View Post
Yeah I've been looking at alternatives too but it's hard to beat the sequence quality and workflow simplicity.

I'll try out a DIY ampliseq approach using NEB enzymes with some modifications:
1. OneTaq Hot Start Master Mix with GC buffer should be sufficient for the multiplex PCR reaction with uracil-containing ampliseq primers. I would scale the reaction down to 20 ul and run a 4/8/16 minute anneal extend. NEB recommends a 68C extension however such a long annealing should provide enough extension activity. Also annoying that Thermo calls their PCR enzyme "AmpliSeq Hifi" when it probably isn't even a high fidelity enzyme.
2. There was a post suggesting using USER and Exonuclease I for the FuPa digest however it was never tested and I'm not sure what the difference is between USER and UDG. I would simply add 10 units of UDG and Endonuclease IV directly to each PCR reaction and incubate at 37C for 1 hour. Adding 3' dA tailing would be convenient for Illumina adapter ligation
3. Blunt/TA Ligase master mix, scaled up to ~ 50 ul though it might be possible to find a 5x mix to keep the ratios as similar to Thermo's as possible. This would work with Ion Xpress adapters or off-the-shelf Illumina barcoded adapters if dA-tailing was performed.
4. Ampure wash, same as Thermo's recommendation but scaled up.
5. Library amp with Q5 Hot start HiFi 2x master mix and P1/X primers or P5/P7 primers.
5. Final size selection and ampure wash as per Thermo's guidelines.

The total cost of the NEB enzymes came out to around $8 per sample. I would order custom P1/X primers from IDT as well as off-the-shelf P5/P7 primers off IDT. Most expensive components would be the AmpliSeq primers followed by the sequencing adapters. The total cost per sample would still be far less than the $100/sample Thermo and Illumina are charging for without the AmpliSeq primers or barcoded sequencing adapters.

Let me know if I'm missing anything.

Ivan
1. are you sure, OneTaq Mix is tolerant to U?
2. I'd add size-select step after 1st PCR to solve primer dimers problem (especially if starting with small amounts of DNA)
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Old 05-22-2018, 09:19 AM   #34
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Quote:
Originally Posted by korostin View Post
1. are you sure, OneTaq Mix is tolerant to U?
2. I'd add size-select step after 1st PCR to solve primer dimers problem (especially if starting with small amounts of DNA)
I checked and OneTaq is compatible with uracil templates. They use it for their bisulfite protocol.

Too bad OneTaq is not a HiFi enzyme. For any liquid biopsy assay where I would want to make calls around 0.1% I would definitely need a HiFi enzyme. Too bad I couldn't find one that is compatible with uracil templates. On the upside OneTaq claims to have 2x higher fidelity than regular Taq.

Size selection after 1st PCR should not be necessary with the FuPa digestion which should digest all primer sequences including template incorporated ones and dimers.

Last edited by idedios; 05-22-2018 at 09:24 AM.
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Old 06-05-2018, 08:30 PM   #35
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Quote:
Originally Posted by HiroMishima View Post
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.
Hi Hiro,

This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.
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Old 06-06-2018, 04:58 PM   #36
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Thank you all the above guys, you provided many informations.

I'm also trying to make my own home-made ampliseq libray kit (for Ion torrent and Illumina)

Based on posts above, I think that the most import thing blongs to :
1) a HIFI DNA polumerase that could tolerrent U in primer( template), there are several candidate: OneTaq (not so HIFI),Phusion U Multiplex PCR Master Mix, KAPA2G FAST Multiplex Kits. I wonder which works best.
2) enzymes that digest primer region and make blunt-end (or single A-tailed) amplicons。 There are many options. UDG & Endo IV might be good choice, because UDG has 100% activity in Endo IV buffer, and Endo IV have both endonuclease and 3'-5' exonuclease activity. However, UDG & Endo IV treated end might be still not able to ligate adaptor. T4 PNK might be needed in this case.


Back to the erly beginning, Do we really need to digest primers? Especially in the conditon that so many tricky details underlines.
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Old 06-06-2018, 05:06 PM   #37
zany
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Quote:
Originally Posted by Buckethead84 View Post
Hi Hiro,

This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.


I think you are right, when you use Endonuclease VIII, you might need T4 pol(or DNA pol1).When you use Endonuclease IV, you might need T4 PNK.

Then the next question is: How to determine concentration of these enzymes?
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Old 06-06-2018, 05:18 PM   #38
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I didn't get information that KAPA 2G could tolerate uracil.

But this one is claimed capable:

KAPA HiFi HotStart Uracil+ ReadyMix (1 x 1.25 mL)

Last edited by zany; 06-06-2018 at 06:41 PM.
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