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Forum: Sample Prep / Library Generation 07-15-2011, 07:35 AM
Replies: 28
Views: 22,234
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 06-30-2011, 06:43 AM
Replies: 28
Views: 22,234
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 04-12-2011, 10:26 AM
Replies: 3
Views: 4,048
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 02-23-2011, 09:11 AM
Replies: 11
Views: 9,693
Posted By elaney_k
Hi BTS, the sequences I think you're referring to...

Hi BTS, the sequences I think you're referring to that are available from Illumina are the new longer indexed adapters used in the TruSeq kits as opposed to the multiplex PCR primers used previously....
Forum: Illumina/Solexa 02-09-2011, 08:34 AM
Replies: 8
Views: 4,980
Posted By elaney_k
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...
Forum: Illumina/Solexa 02-09-2011, 07:54 AM
Replies: 1
Views: 3,065
Posted By elaney_k
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...
Forum: Illumina/Solexa 01-26-2011, 03:28 AM
Replies: 24
Views: 11,847
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 12-23-2010, 05:40 AM
Replies: 8
Views: 15,993
Posted By elaney_k
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...
Forum: Illumina/Solexa 12-23-2010, 05:31 AM
Replies: 1
Views: 2,917
Posted By elaney_k
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...
Forum: Illumina/Solexa 04-30-2010, 09:55 AM
Replies: 28
Views: 12,991
Posted By elaney_k
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...
Forum: Core Facilities 04-26-2010, 08:39 AM
Replies: 8
Views: 6,007
Posted By elaney_k
How secure is data that's uploaded through the...

How secure is data that's uploaded through the Galaxy interface?
Forum: Illumina/Solexa 03-22-2010, 05:22 AM
Replies: 2
Views: 3,034
Posted By elaney_k
I reckon it's adapter dimer products related to...

I reckon it's adapter dimer products related to the amount of adapter you used in the ligation step. I generally dilute the adapter 1:30 for use in the ChIP-seq prep protocol which stopped any...
Forum: General 03-04-2010, 04:26 AM
Replies: 4
Views: 2,054
Posted By elaney_k
might not be such a mad idea if they were looking...

might not be such a mad idea if they were looking to use a different platform like Illuminas new HiSeq 2000. I think the type of project you're talking about would be significantly cheaper on that...
Forum: Illumina/Solexa 02-22-2010, 06:06 AM
Replies: 9
Views: 14,492
Posted By elaney_k
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...
Forum: Illumina/Solexa 01-15-2010, 09:52 AM
Replies: 2
Views: 1,499
Posted By elaney_k
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...
Forum: Illumina/Solexa 01-11-2010, 02:29 AM
Replies: 8
Views: 4,158
Posted By elaney_k
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...
Forum: Illumina/Solexa 11-03-2009, 10:28 AM
Replies: 9
Views: 5,812
Posted By elaney_k
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...
Forum: Illumina/Solexa 11-03-2009, 08:16 AM
Replies: 9
Views: 5,812
Posted By elaney_k
Hi Andi, Does Bowtie allow you to align...

Hi Andi,

Does Bowtie allow you to align reads to a reference that's shorter than your read length?
Forum: Illumina/Solexa 11-03-2009, 03:49 AM
Replies: 9
Views: 5,812
Posted By elaney_k
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...
Forum: Illumina/Solexa 10-30-2009, 04:53 AM
Replies: 9
Views: 5,812
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 10-16-2009, 08:19 AM
Replies: 8
Views: 15,993
Posted By elaney_k
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...
Forum: Illumina/Solexa 10-08-2009, 03:14 AM
Replies: 25
Views: 10,742
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 10-08-2009, 02:15 AM
Replies: 8
Views: 15,993
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 10-07-2009, 07:49 AM
Replies: 9
Views: 7,933
Posted By elaney_k
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...
Forum: Sample Prep / Library Generation 10-07-2009, 04:36 AM
Replies: 1
Views: 3,177
Posted By elaney_k
Can you post a picture of your post enrichment...

Can you post a picture of your post enrichment PCR products run on a gel?
Showing results 1 to 25 of 55

 


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