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sleeplessstudent 05-10-2013 11:01 AM

Library sequencing
I am planning on performing an illumina multiplex sequencing run on a library of sequences that I have compiled. They are digested out of plasmid and consist of ~10 unvaried nucleotides, 60 varied nucleotides, ~40 unvaried nucleotides, 60 varied nucleotides, then 10 unvaried nucleotides in that order. I have seen in previous papers that there are some issues with the cluster identification step for sequences which are unvaried in their first few nucleotides. Is this still a major problem? Is there a way to get around this without adding extra varied nucleotides to the ends of my sequences? Or is this the only robust solution?

kwaraska 05-10-2013 12:23 PM

You need to design a custom sequencing primer.

Use the sequence of the adapter+sequence of your non-variable region(10 bases)

Then create a sequencing primer that is complementary to the last base of your nonvariable region-then work back.

When the Tm of the primer is above 65 (that way it will work on both HiSeq and MiSeq) you have that primer synthesized and HPLC purified.

Use the primer at 100uM.

Whoever is doing you sequencing will know how to use it.

SNPsaurus 05-10-2013 12:51 PM

If you are sequencing a small number of plasmid-derived inserts, then you are likely to not need the full number of reads in a lane. An alternative to creating a custom primer is to just spike your library into a control lane of PhiX on a HiSeq, or mix in a higher-complexity library (usually someone has something they want a little more sequence for) on a MiSeq. It seems like the recent software changes have made the MiSeq less sensitive to complexity reductions anyway.

tambo 05-13-2013 11:00 PM

Please can your help in any library for plasmodium gametocytes. Thnaks . Erny

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