short sequencing reads

vlee2 · 02-21-2011, 06:30 AM

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.

Thanks!

flxlex · 02-22-2011, 05:54 AM

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...

vlee2 · 02-22-2011, 06:10 AM

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.

Thanks

pmiguel · 02-23-2011, 07:11 AM

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.

--
Phillip

Soulbee · 02-28-2011, 08:34 AM

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?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Genome Res De novo bacterial genome sequencing: millions of very short reads assembly	b_seite	Literature Watch	1	10-05-2017 12:26 AM
PubMed: Local De Novo Assembly of RAD Paired-End Contigs Using Short Sequencing Reads	Newsbot!	Literature Watch	0	05-06-2011 12:40 AM
PubMed: PerM: Efficient Mapping of Short Sequencing Reads with Periodic Full Sensitiv	Newsbot!	Literature Watch	0	08-14-2009 06:00 AM
PubMed: Mapping accuracy of short reads from massively parallel sequencing and the im	Newsbot!	Literature Watch	0	07-29-2009 06:00 AM
Sequencing of natural strains of Arabidopsis thaliana with short reads.	strob	Literature Watch	0	01-21-2009 07:19 AM

02-22-2011, 05:54 AM	#2
flxlex Moderator Location: Oslo, Norway Join Date: Nov 2008 Posts: 415	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...

02-22-2011, 06:10 AM	#3
vlee2 Member Location: Richmond, VA Join Date: Apr 2009 Posts: 19	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. Thanks

02-23-2011, 07:11 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	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. -- Phillip

02-28-2011, 08:34 AM	#5
Soulbee Member 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?