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Old 06-15-2008, 09:32 PM   #1
satp
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Default Question about Sequencing of mixed small RNA cDNA library

Hi everyone,

I want to sequence two small RNA libraries by Solexa, and don't need very deep coverage. So I want to mix these two libraries in one run. I knew from this literature that we can add indexing nucleotides to the individual cDNA libraries so that the origin of the sequences can be traced. But that literature didn't tell me how to do. Is anyone here has sequenced the mix small RNA libraries by Solexa? I wonder where to add the indexing nucleotides, 3' adaptor or 5' adaptor? And whether should I change the sequence of RT, PCR and sequence primer.
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Old 06-19-2008, 01:56 AM   #2
james hadfield
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Default RE:Default Question about Sequencing of mixed small RNA cDNA library

Hi SATP,
The indexing on Illumina can be done in two ways.
1: add an index sequence, e.g. 4bp, to the 3' end of the 5' adapter molecule immediately after the sequencing oligo binding site. This is pretty easy to do but uses four of your best quality sequences. It can also be tricky to strip the adapter molecules from the sequence afterwards if you are doing 36bp runs as there is not enough sequence to definitively identify adapter.

2: add a second sequencing primer site and index sequence on the 5' end of the 3' adapter molecule. This allows a second sequencing primer hybridisation and sequencing run, it is more complex and requires paired end hardware, but generates high quality index sequence as well as high quality insert sequence.

This work has now been submitted for publication.

Last edited by james hadfield; 06-14-2009 at 12:23 AM. Reason: update
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Old 06-19-2008, 05:59 AM   #3
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I get it. Thanks for your help.
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