Forum: Sample Prep / Library Generation
07-15-2011, 06:35 AM
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Replies: 28
Views: 22,568
Hi Dan,
Sincere apologies for such a late...
Hi Dan,
Sincere apologies for such a late response! We used 50ng of globin reduced total RNA as input for the Ovation RNA-seq protocol and 2ug of total RNA as input for the Illumina protocol...
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Forum: Sample Prep / Library Generation
06-30-2011, 05:43 AM
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Replies: 28
Views: 22,568
Hi,
I've used NuGEN RNA-seq sample prep...
Hi,
I've used NuGEN RNA-seq sample prep reagents and would never recommend them to anyone. The resulting data looked awful with far more reads mapping to intronic regions over exonic regions. The...
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Forum: Sample Prep / Library Generation
04-12-2011, 09:26 AM
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Replies: 3
Views: 4,121
Tbh if you're going that far with the protocol...
Tbh if you're going that far with the protocol I'd suggest buying custom-made Illumina barcoded adapters and PCR enrichment primers and finishing the sample prep yourself, I've pooled RNA-seq samples...
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Forum: Sample Prep / Library Generation
02-23-2011, 08:11 AM
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Replies: 11
Views: 9,861
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Forum: Illumina/Solexa
02-09-2011, 07:34 AM
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Replies: 8
Views: 5,068
Hi Andrew,
I think you're referring to the...
Hi Andrew,
I think you're referring to the Illumina Multiplexing protocol where the index is introduced during the enrichment PCR stage of the protocol. If you contact Illumina techsupport...
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Forum: Illumina/Solexa
02-09-2011, 06:54 AM
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Replies: 1
Views: 3,132
Hi Bruce,
We've done many of these types of...
Hi Bruce,
We've done many of these types of experiments using custom-made indexed adapters (different to the new Illumina indexed adapters in their TrueSeq kits) and SR or PE sequencing. We didn't...
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Forum: Illumina/Solexa
01-26-2011, 02:28 AM
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Replies: 24
Views: 12,085
Hi,
I've managed to sequence samples for a...
Hi,
I've managed to sequence samples for a collaborator on the Illumina GA that were target-enriched using the nimblegen/454 prep. The input DNA had the 454 sequencing adapters on either end of...
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Forum: Sample Prep / Library Generation
12-23-2010, 04:40 AM
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Replies: 8
Views: 16,046
Hi guys,
Shameless plug I know but we...
Hi guys,
Shameless plug I know but we finally managed to publish the multiplex target enrichment study for anyone that's still interested in it :)
Multiplex Target Enrichment Using DNA Indexing...
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Forum: Illumina/Solexa
12-23-2010, 04:31 AM
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Replies: 1
Views: 2,960
Hi Marineman,
Have you tried to do a second...
Hi Marineman,
Have you tried to do a second gel isolation step where you cut out only the 110bp fragment of your library? You will loose a certain percentage of your library but should get rid of...
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Forum: Illumina/Solexa
04-30-2010, 08:55 AM
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Replies: 28
Views: 13,135
I think earlier versions of the protocol used...
I think earlier versions of the protocol used Ambions RNA Fragmentation Reagents (AM8740). It's a 10x solution so would assume you could just dilute it down... no guarantee it's still the same one...
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Forum: Core Facilities
04-26-2010, 07:39 AM
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Replies: 8
Views: 6,066
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Forum: Illumina/Solexa
03-22-2010, 04:22 AM
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Replies: 2
Views: 3,100
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Forum: General
03-04-2010, 03:26 AM
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Replies: 4
Views: 2,103
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Forum: Illumina/Solexa
02-22-2010, 05:06 AM
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Replies: 9
Views: 14,575
Hi guys,
The cluster...
Hi guys,
The cluster seperation/identification is carried out in the first 4 cycles of sequencing for SCS 2.6, I think previously it was the first 2 cycles, so if all your samples contain the same...
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Forum: Illumina/Solexa
01-15-2010, 08:52 AM
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Replies: 2
Views: 1,535
I'm not sure if I understand your question,...
I'm not sure if I understand your question, however for detection of A to I RNA editing you need to sequence both the genomic DNA and RNA. If editing occurs you will only see an "A" in the DNA...
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Forum: Illumina/Solexa
01-11-2010, 01:29 AM
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Replies: 8
Views: 4,225
Hi ikrier,
Whilst you renamed the header for...
Hi ikrier,
Whilst you renamed the header for the sequence data line "@SOLEXAWS1_2062VAAXX:1:1:46:643#0/1" you didn't rename the header for the quality scores. This is what an input file for eland...
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Forum: Illumina/Solexa
11-03-2009, 09:28 AM
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Replies: 9
Views: 5,901
Hi Andi,
I was initially looking through...
Hi Andi,
I was initially looking through data from a ChIP-seq run that had a really low % alignment and by eye I could see the same sequence repeated, when i checked the sequence it turned out to...
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Forum: Illumina/Solexa
11-03-2009, 07:16 AM
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Replies: 9
Views: 5,901
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Forum: Illumina/Solexa
11-03-2009, 02:49 AM
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Replies: 9
Views: 5,901
Hi Andi,
I look for a slightly truncated...
Hi Andi,
I look for a slightly truncated adapter sequence in the export files using the grep command so for ChIP-seq (single read adapters) the command I use is:
grep -c...
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Forum: Illumina/Solexa
10-30-2009, 03:53 AM
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Replies: 9
Views: 5,901
Hi Andibody,
What type of preps are you...
Hi Andibody,
What type of preps are you doing? I've seen it with Chip-seq and mRNA-seq preps were further analysis of the sequenced reads showed a percentage of adapter-dimers in the samples. This...
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Forum: Sample Prep / Library Generation
10-16-2009, 07:19 AM
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Replies: 8
Views: 16,046
Thanks guys!
I quantified the samples using...
Thanks guys!
I quantified the samples using a qubit flourimeter (Invitrogen) as it can quantify dsDNA even in the presence of ssDNA e.g. primers. I wouldn't recommend a nanodrop or bioanalyzer for...
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Forum: Illumina/Solexa
10-08-2009, 02:14 AM
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Replies: 25
Views: 10,919
These guys seem to have come across the same...
These guys seem to have come across the same problem: http://seqanswers.com/forums/showthread.php?t=2582
I have also performed a second gel-isolation of some of my samples post enrichment PCR...
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Forum: Sample Prep / Library Generation
10-08-2009, 01:15 AM
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Replies: 8
Views: 16,046
Hi guys,
I've enriched a set of 9 indexed...
Hi guys,
I've enriched a set of 9 indexed samples using 1 Agilent sureselect assay. I got very even representation of the indexed samples in the final sequenced data (9.4-11.7% representation of...
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Forum: Sample Prep / Library Generation
10-07-2009, 06:49 AM
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Replies: 9
Views: 8,004
There's a really great paper that came out from...
There's a really great paper that came out from the sanger Institute (is that near you?), "A large genome center's improvements to the Illumina sequencing system" PMID 19034268 which addresses the...
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Forum: Sample Prep / Library Generation
10-07-2009, 03:36 AM
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Replies: 1
Views: 3,260
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