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Old 08-16-2012, 04:19 PM   #1
owik303
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Question Amplicon Sequencing with Ion Torrent

Hi, I've been reading threads in this forum for months now and have found a lot of useful information, and seen many questions answered and figured id give it a try.

I am currently working on multiplexing 188 bar coded amplicon samples into a single mix and sending out an equal molar mixture for ion torrent sequencing to a third party vendor.

This will be my 9th mixture for ion torrent sequencing and recently (projects 7 and 8) my results have been diminishing in quality. A closer look at the sequencing data shows that a surprisingly larger number of short reads (under 60 bp's). This is strange because I have been using Ampure XP beads to purify my samples before mixing them together and see little to no DNA of that size present on gels. The weirdest thing is that I ran out the final mixture of the last 5 of my projects on a single gel, quanted the area below 100 bp's and there appears to be negligible difference in DNA concentration between mixes, while the sequencing data varies greatly.

My purification protocall has remained the same through all 5 projects (use a 1:1 ratio of bead to PCR product, wash 2x with 70% etoh elute in the same volume of TE ph 7.5) as well as my PCR cycling conditions, reagents, consumables ect...

The concerning difference that comes to mind is that originally I was using the 314 ion chip for the first few projects and recently switched to the 316 ion chip, regardless of chip size I requested the run to be done with the 100 bp ion kit.

Further analysis of the worst and most recent sequencing run showed that a smaller but decent % of the sequences was the 5p amplicon primer and associated bar code, which is what immediately fallows the ion A adapter sequence and key.

I dont understand how this could have made it through emulsion PCR, or been captured and sequenced seeing as it only had the Ion A adapter sequence present and NOT the Ion P1 adapter sequence. Has anyone elce encountered this problem? I feel like Ion torrent is changing their sequencing protocol and not informing the public, maybe im just paranoid and missing something simple... Please fell to give input and/or rant
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Old 08-20-2012, 06:56 AM   #2
IonTorrent
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Hi there,

Indeed, this does seem unusual and I'd like to help you with your question. I tried to send you a follow up note but your profile settings do not seem to allow receipt of messages from non-contacts.

Quote:
Originally Posted by owik303 View Post
Hi, I've been reading threads in this forum for months now and have found a lot of useful information, and seen many questions answered and figured id give it a try.

I am currently working on multiplexing 188 bar coded amplicon samples into a single mix and sending out an equal molar mixture for ion torrent sequencing to a third party vendor.

This will be my 9th mixture for ion torrent sequencing and recently (projects 7 and 8) my results have been diminishing in quality. A closer look at the sequencing data shows that a surprisingly larger number of short reads (under 60 bp's). This is strange because I have been using Ampure XP beads to purify my samples before mixing them together and see little to no DNA of that size present on gels. The weirdest thing is that I ran out the final mixture of the last 5 of my projects on a single gel, quanted the area below 100 bp's and there appears to be negligible difference in DNA concentration between mixes, while the sequencing data varies greatly.

My purification protocall has remained the same through all 5 projects (use a 1:1 ratio of bead to PCR product, wash 2x with 70% etoh elute in the same volume of TE ph 7.5) as well as my PCR cycling conditions, reagents, consumables ect...

The concerning difference that comes to mind is that originally I was using the 314 ion chip for the first few projects and recently switched to the 316 ion chip, regardless of chip size I requested the run to be done with the 100 bp ion kit.

Further analysis of the worst and most recent sequencing run showed that a smaller but decent % of the sequences was the 5p amplicon primer and associated bar code, which is what immediately fallows the ion A adapter sequence and key.

I dont understand how this could have made it through emulsion PCR, or been captured and sequenced seeing as it only had the Ion A adapter sequence present and NOT the Ion P1 adapter sequence. Has anyone elce encountered this problem? I feel like Ion torrent is changing their sequencing protocol and not informing the public, maybe im just paranoid and missing something simple... Please fell to give input and/or rant

Last edited by IonTorrent; 08-20-2012 at 07:00 AM.
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Old 08-20-2012, 08:18 AM   #3
owik303
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Thanks for responding and I wold love to hear what you have to say, I have updated my profile to receive emails from everyone now, please email away! If for whatever reason you still encounter a problem I would be more than happy to supply you with my direct email.
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Old 04-19-2016, 02:07 PM   #4
mhgseq
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Have you solve your probelm of <60bp short reads.

I have similar problem with my fusion primer amplicon seq with Ion Torrent. Everything is fine in last several month with up to 16 barcode. When I switch to 50+ barcode, we encounter lots of short reads. We are using 318 chip.
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Old 04-19-2016, 02:59 PM   #5
owik303
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mhgseq - we have resolved this issue greatly, I have demonstrated on the same library short reads cannibalizing up to 70%+ of the total reads form a 314, 316 and 318 chips even with deceased loading concentrations than recommended. The same library if heated to 95c for 5 min then slow cooled 0.1 degree / second to 37 (heat cooled as we call it) before being diluted and added to the ISP for emPCR dropped short reads to under 10% of the total. We believe this was due to aggeration of amplicons as our insert size is ~ 100 bases with ~25 bases of fixed primer region on both the 5' and 3' end. The reason this affects amplicon sequencing so much more is due to the majority of the library having identical fixed regions and limited diversity between them, which is not typically the case in genomic libraries. These aggerates carry through template dilution and emPCR seeding as its all done at 25c. This results in high polyclonal reads, high # of empty wells, high low quality reads and typically a larger # of what appear as truncated reads.
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Old 04-19-2016, 03:46 PM   #6
mhgseq
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Thank you so much for the info. I am also do the similar short amplicon ( ~100bp + primers+ adaptor). But for the loading, we load much higher concentration for OT2 (200 pM vs the recommend 26 pM), I get 8% empty well and 21% polyclonal.
I am thinking about reducing the loading concentration as you mentioned above. I will certainly try your heat cooled approach.
Do you do the heat cooled before purification or after?
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Old 04-19-2016, 03:51 PM   #7
owik303
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we were loading less than 26pM routinely as a way to combat the affect of the aggerates, to as little as 10pM, this helped with polyclonal but had no affect on the short read fragments. The heatcool should be performed on the purified library after it has been quantified with picogreen (not bioanalyzer as the aggeration alters the size it measures and messes up your concentration calculation) and you are preparing to make the dilutions necessary to get your library to a concentration where there is 1 copy per ISP for emPCR. so heat cool right before emPCR tempalting, also the longer you let you emPCR rxn sit around after its complete (i believe ion says its good up to 1 week) the worse your data from sequencing will be, at least it was for us.
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Old 04-20-2016, 08:16 AM   #8
mhgseq
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We do check the bioanalyzer, not a lot of large size shift. But we do see a lots of small variation in size (our amplicon is about 200bp (include primer and adaptor), we do see peak at 180-250bp in bioanalyzer, occasionally see 300-400bp).

I checked with IonTorrent support, they think my 21% polyclonal is underestimated. According to their suggestion, I will change trim parameter to reanalyze it. I will update how the result looks like.
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Old 04-28-2016, 07:36 AM   #9
mhgseq
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thanks a lot. Your heat cooled approach works well. We get much better reads this time.
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