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Old 12-02-2016, 02:27 AM   #1
neko_33
Junior Member
 
Location: Tokyo

Join Date: Dec 2016
Posts: 3
Default Terrible overclustering -- 16S EMP protocol

Hi SEQ-users,

We've been using the following protocol for 16S V4 region amplicon sequencing on MiSeq, which I think is very familiar to many of you:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400413/

For several years, most members in our lab have been constantly successful in that method.
Recently, however, cluster density is abnormally high -- about 1300~1500K/mm2(v2 kit) -- and sequencing quality very low. We tried over and over, reducing the library concentration from 15pM to 10pM, renewing sequencing primers, renewing phiX controls, but none of these worked.

In additon, libraries prepared with other methods (e.g. 2-step PCR, NEB UltraDNA Library Prep Kit, bacterial RNA-seq, etc.) are still sequenced fairly well. This probably means that, our trouble is not due to the MiSeq instrument or our poor sample handling.

Do any of you have such problems?
Or, does anyone knows the solution?

Thanks a lot in advance.
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