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Old 06-27-2010, 01:22 AM   #1
Khen Khermesh
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Location: Israel

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Question Massive parallel amplicon sequencing on Illumina GA

My questions are about amplicon sequencing.
I would like to sequence numerous PCR products on the Illumina GA.
I have about 50 amplicons that are my test group which I plan to scan on a set of 5 different RNA samples resulting in ~ 250 PCR products (amplicons).

1. Is there a recommended protocol for the preparation of the amplicons for sequencing on the Illumina GA?
2. Can I add the adpters directly to the PCR primers used to generate the amplicons?
3. What is the best way to barcode (tag) the different PCR amplicons coming from different RNA sample?
Best
Khen
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Old 06-28-2010, 12:48 PM   #2
cliffbeall
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1. I checked in to doing amplicon sequencing with Illumina and as far as I can tell it has not been tried much
2. You would want to get 5' phosphorylated primers to ligate to the Illumina adapters
3. Illumina sells barcoded adapter kits, or you could append homebrewed barcodes to the PCR primers. The Illumina system uses a separate read just for the barcode.

You will likely see amplification biases, so it probably won't be a great comparison between transcripts, but should be ok between samples.
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Old 06-28-2010, 02:24 PM   #3
454newbie
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Contact Andy May at fluidigm: [email protected]. His group have this worked out.

Last edited by 454newbie; 03-08-2011 at 10:28 AM.
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Old 07-09-2010, 06:44 AM   #4
adamdeluca
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Quote:
Originally Posted by Khen Khermesh View Post
2. Can I add the adpters directly to the PCR primers used to generate the amplicons?
This will limit you to only sequencing the length of your reads into the amplimer. If you sonicate the PCR products before ligating the adaptors you can get coverage in the center of the amplimer.

http://www.cofactorgenomics.com
Did an Illumina amplicon run for us.

Given the high error rate of 454 sequencing, I think the sonicated amplimer + short read sequencing is the way to go.
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