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Old 02-21-2011, 05:30 AM   #1
Location: Richmond, VA

Join Date: Apr 2009
Posts: 19
Default short sequencing reads

We have a short reads problem when sequencing previously successfully sequenced library. Could somebody help me to figure out our problem?
Img1.png represent our first sequencing run of this library. Then at the same time we prepared two LV emPCR for this library. Img2.png represent our second sequencing run using beads from one LV emPCR preparation above and img3.png - third sequencing run using beads from another LV emPCR preparation.
Where these huge number of small fragments came from? 99% of reads carry same adapters of this library, so its not some sort of contamination.

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Old 02-22-2011, 04:54 AM   #2
Location: Oslo, Norway

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Did you store the enriched beads for the third run for some time before putting them on the plate? Did the emulsion oil have a different lot number for the third run? Just some thoughts...
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Old 02-22-2011, 05:10 AM   #3
Location: Richmond, VA

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Thanks for your reply flxlex!
I stored those beads for the 3rd run about 4 days. We also checked the storage stability of enriched DNA beads and 3 months old beads produced good results.
We made beads for the 2nd and 3rd run at the same time, so we used same lot number of oil, and reagents.
Is there a possibility of some glitch on the sequencing side, because there was nothing wrong with beads preparation? Though another region on the plate had normal size distribution.

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Old 02-23-2011, 06:11 AM   #4
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Location: Purdue University, West Lafayette, Indiana

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Did you anneal the sequencing primer before storing the enriched beads? If so, maybe there was some primer denaturation over the 4 days the beads were waiting. Then signal from the beads might have been weak, resulting in short reads.

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Old 02-28-2011, 07:34 AM   #5
Location: Toronto, Canada

Join Date: Jun 2010
Posts: 25

it looks like not very much of your actual library has amplified which makes me wonder if your adaptors/primers are annealed properly.. how was your enrichment?
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