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-   -   custom sequencing primers (http://seqanswers.com/forums/showthread.php?t=2160)

athos 07-23-2009 03:07 AM

custom sequencing primers
 
Hi,

Does anyone use their own primers for sequencing on the GAII, i.e rather than the Illumina supplied primer? If so, what purification/modifications do you use? I'm wondering if, for example, any phosphorothioate bonds would be necessary... any thoughts appreciated!

Thanks.

ScottC 07-28-2009 05:26 PM

I don't think the sequencing primers are modified. The sequences are freely available from Illumina if you ask (they may even be posted here somewhere).

Cheers,

Scott.

james hadfield 08-03-2009 04:13 AM

The sequencing primers are indeed unmodified. We order PAGE purified although I am not sure there is a requirement for this. If you want to know the sequences I would suggest contacting Illumina techsupport directly and asking for the latest release of their "sequence information for customers" pdf.
This way there should be no confusion.
James.

ovallon 02-04-2011 05:59 AM

a sequencing primer OTHER than the Illumina primer
 
Hi !
My first post here :)
Quote:

Originally Posted by athos (Post 6780)
Hi,

Does anyone use their own primers for sequencing on the GAII, i.e rather than the Illumina supplied primer?

Possibly the same question that you had in mind, but phrased differently.

What I would like to know is whether it is possible, when you are doing amplicon sequencing, to use a primer WITH A SEQUENCE DIFFERENT from that used by Illumina.
Let me explain. Imagine you have amplified a region, say the flanking sequences of a transposon, and made an Illumina library that has the Illumina primers at both ends. But you don't want to sequence the region immediately downstream of the primer, because you already know what it is. You want to start sequencing WITHIN the insert.
Can you prime sequencing with an oligonucleotide that anneals to a common part of all your inserts, downstream of the adapter ?

If yes, must you use the same sequencing primer for the whole chip, or can you use different sequencing primers in different lanes ?

Final question: is it true that identification of the clusters is hampered when the first nucleotides of all (or most of) the sequences are identical ? This would be the case for example if you were sequencing through polyA-tails, or through a region that is common to all your inserts. I have heard that it is wise in this case to add 2 Ns right after the sequencing primer to make sure that sequences differ locally, allowing pixels to be grouped into clusters. True ?

HESmith 02-04-2011 07:58 AM

1. You can use custom primers for sequencing. The Tm should be similar to the standard primer.

2. You can use different primers in different lanes of the flow cell.

3. Biased nucleotide composition in the first few cycles (I believe this number varies with different software versions) adversely affects cluster calling.

-Harold

fkrueger 02-04-2011 10:47 AM

It is indeed true that low-diversity at the start of sequences will cause problems with cluster identification. See this thread.


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