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Old 08-20-2012, 07:56 AM   #2
Location: Guilford, CT and S.F., CA

Join Date: Jan 2010
Posts: 64

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.

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 08:00 AM.
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