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Old 09-06-2018, 08:47 AM   #1
bheida
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Default Custom Sequencing Primer

Hi,

I am writing my thesis and I ran into a question. Maybe someone here happens to know the answer to this.

We have an Illumina Miseq in our lab. For our sequencing approach we used custom sequencing primer (well 18 and 20 on the MiSeq cartridge). I know that these primers are used during the sequencing process itself. But are they also used for the bridge amplification that precedes the actual sequencing? Or does the polymerase for the bridge amplification start directly at the Illumina adapter and the flow cell.

I hope I phrased this clearly enough. Any insight would be much appreciated.

Thanks in advance.
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Old 09-06-2018, 11:24 PM   #2
GSviral
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Hi bheida,

This is a good question. I may be a bit rusty on my theory but I can try to help.

I believe when the libraries have hybridised to the flow cell via the P5 and P7 oligos the complementary strands are synthesised and the original template molecules are washed away. Then the bridge amplification occurs, the 3-prime P5/P7 end forms the bridge with complementary oligo on the flow cell, and the new strand is synthesised using that flow cell oligo as the primer.

So I think the bridge amplification begins at the flow cell adapter/oligo. The sequencing primers are used during actual read synthesis.

Someone else can correct me if I am wrong, it's been a while since I've thought about this!
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Old 09-07-2018, 07:06 AM   #3
misterc
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GSviral, you're correct on all statements there
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Old 09-09-2018, 04:05 AM   #4
bheida
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Thank you guys very much for your help. It can be difficult to find this type of information anywhere else on the web.
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