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Old 02-04-2011, 05:59 AM   #4
ovallon
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Location: France

Join Date: Feb 2011
Posts: 1
Default a sequencing primer OTHER than the Illumina primer

Hi !
My first post here
Quote:
Originally Posted by athos View Post
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 ?
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