SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Multiplex on HiSeq? cbrennan Illumina/Solexa 9 03-24-2011 11:51 AM
problem with mRNA multiplex libraries sc10021 Illumina/Solexa 8 01-27-2011 02:30 PM
Tags in Multiplex suludana Illumina/Solexa 2 12-01-2009 06:59 AM
Using some of the 12 multiplex tags bioinfosm Illumina/Solexa 2 08-04-2009 10:52 PM
Multiplex suludana Illumina/Solexa 0 01-13-2009 03:30 AM

Reply
 
Thread Tools
Old 06-26-2009, 02:45 PM   #1
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,173
Default A possible problem with multiplex tags just occurred to me

We just completed a run where an interesting artifact of GA sequencing surprised us a little. A researcher wanted to sequence a reduced representation of a genome. He digested genomic DNA with AluI, ran the digest out on a gel and isolated fragments in a particular size range. That DNA was prepared for sequencing without further fragmentation. This means that the 5' end of all the DNA strands started with 'CT'. The consequences of this did not occur to us until we saw the images of the first cycle of sequencing. The extreme bias in the base composition of the first two cycles completely flumoxed the cluster identification routines of the Illumina image analysis software. No problem though as I can rerun the analysis from scratch with the pipeline but only using cycles 3-36.

This got me thinking. Suppose you have designed your own multiplex adapter oligos, placing the bar code immediately downstream of the primer site. Depending on the base representation in the mixture of bar codes you are using this could lead to a problem like the one we encountered above, but you can't simply ignore those cycles since you will need those bases to sort the samples.

Have I made any sense at all? Has anyone encountered this situation using bar codes or have you developed protocols to make sure you avoid it?
kmcarr is offline   Reply With Quote
Old 06-29-2009, 08:27 AM   #2
swbarnes2
Senior Member
 
Location: San Diego

Join Date: May 2008
Posts: 912
Default

I thought that was the purpose of the control lane; that even if your other lanes had weird biases, that the software would trust that those lanes were correct if the control lane was showing the right ratio of letters.
swbarnes2 is offline   Reply With Quote
Old 06-29-2009, 12:25 PM   #3
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,173
Default

Yes, the control lane can be used exclusively to calculate the crosstalk matrix and phasing numbers. But there was a significant problem with the cluster identification. The cluster template is determined in the first three cycles (with the new, v2.4 software, prior to that it was just the first two cycles) and I was told by the tech support person that the software needs to be seeing some signal in all four channels to identify cluster locations. That was my main concern about samples in which (nearly) all of the fragments have identical bases for the first cycle or two.

However I have now found that in addition to 5' ends of all the fragments being identical the flow cell was horribly overloaded, making it impossible for the software to isolate clusters even if the base composition was perfectly balanced. I guess we'll be rerunning the sample at a much lower concentration and I'll see then what effect the biased 5' ends have on cluster identification.
kmcarr is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 07:16 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO