3 replies to this topic

[hbn]

I'm looking at the Illumina protocol (I've attached the protocol) for multiplexed sequencing on the GAIIx (Genome Analyzer, NGS).

I was wondering; two different adapters are to be ligated on both ends of the fragments, but how can you achieve this? I guess if you're using two different adapters (let's call them adapter1 and adapter2), some fragments will have adapter1 on both ends, some have adapter2 on both ends and some will have the two different adapters on both ends. But how do they make sure / select for products with different adapters at both ends?
I really hope someone can explain this to me...

They are performing a PCR amplification, but I guess that if you have two of the same adapters, you could get a product with two forward primers or two reverse primers...

Edited by hbn, 02 March 2011 - 07:57 AM.

[Maddie]

Have you seen what the adaptors look like? They form a Y and this Y contains both adaptor 1 and 2 because it's double stranded. Hmm it's hard to explain. A figure will speak for itself. See attached file.

Attached Files

•  great fig sequences adap&primers.pdf   24.43K   209 downloads

[hbn]

Maddie, on 02 March 2011 - 12:07 PM, said:

Have you seen what the adaptors look like? They form a Y and this Y contains both adaptor 1 and 2 because it's double stranded. Hmm it's hard to explain. A figure will speak for itself. See attached file.

Heey Maddie,
Thanks for the explanation and the PDF, it's quite clear. It's a complex system, but it's pretty smart...

[Maddie]

hbn, on 02 March 2011 - 11:41 PM, said:

Maddie, on 02 March 2011 - 12:07 PM, said:

Have you seen what the adaptors look like? They form a Y and this Y contains both adaptor 1 and 2 because it's double stranded. Hmm it's hard to explain. A figure will speak for itself. See attached file.

Heey Maddie,
Thanks for the explanation and the PDF, it's quite clear. It's a complex system, but it's pretty smart...

It sure is. I'm so excited to play with NGS. I hope you get gigas of kb