I have a researcher who wishes to do targeted resequencing of a ~100kb
region from 100-200 individuals. We are planning to generate the targeted
DNA by pooling of tiled long-range PCR products. Obviously the amount if
sequence generated in a single lane of the GA flow cell is sufficient to
cover many individuals. I was hoping to use bar coding of the individual
samples (exactly like the 454 MID tag procedure) to multiplex samples in
each lane of flow cell. I have heard tell that there are people out there
doing this with Illumina but my searches for specifics have thus far proved
fruitless.
Do any of you good folks have any experience or protocols you might like to
share for multiplexing samples by bar code in an Illumina GA run.
Thank in advance,
Kevin M. Carr
**************************
Bioinformatics Specialist
Research Technology
Support Facility
202-D Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824
Ph: (517) 353-6794
Fax:(517) 353-8638
**************************
Thanks for posting this; in many ways it is very similar to what our
researcher here wants to do so will be very helpful. After reading the
paper I have a couple of questions if you don't mind.
First, you used tiled PCR to cover the cp genome which is what we suggested
to our researcher to cover the 100kb region he is interested. Given the
disparity in coverage you observed for the various amplicon tiles would you
now choose a different method; for example a DNA capture array?
Second, your supplementary material shows that you modified the adapters for
both ends. I may be missing something but why modify the "1" adapters (e.g.
CCT1, GGT1). These end up at the other end of the DNA strand from the
sequencing primer so it seems to me that a common adapter for this end
should be fine. Am I not understanding the library preparation protocol for
the Illumina properly?
Finally, you showed that you can get misincorporations in your index
sequences. Given the sequences you chose it appears that you could
misassign reads if there is a particular misincorporated base. For example,
in the MPLX S1 sample two of the indexes you used were AAT and ATT; an A->T
substitution in the first case or T->A in the second would change the
assignment of that read. It seems that there isn't any way to detect and
correct these. Did you just accept that this may happen but that the
frequency was low enough to ignore?
Thanks,
Kevin M. Carr
**************************
Bioinformatics Specialist
Research Technology
Support Facility
202-D Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824
Ph: (517) 353-6794
Fax:(517) 353-8638
**************************
> From: Aaron Liston <lis...@science.oregonstate.edu>
> Reply-To: Solexa User Group <sol...@googlegroups.com>
> Date: Wed, 27 Aug 2008 10:53:52 -0700 (PDT)
> To: Solexa User Group <sol...@googlegroups.com>
> Subject: Re: Multiplexing in a flow cell lane by bar coding
>
>
The sound you hear is me slapping my head and grunting a Homer Simpsonesque
DOH!.
The fact is I had not completely grokked the nature of the Illumina "Y"
adapters for library preparation. The tumblers finally clicked, thanks.
Thanks also for your answers to the other questions.