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Search: Posts Made By: james hadfield
Forum: Introductions 11-11-2015, 01:12 PM
Replies: 1
Views: 934
Posted By james hadfield
Hi Kurt, Please get in touch directly....

Hi Kurt,
Please get in touch directly. [email protected], @CIgenomics, or http://core-genomics.blogspot.co.uk.
I'd like to find out more and will take a look.
James.
Forum: Sample Prep / Library Generation 11-11-2015, 01:06 PM
Replies: 3
Views: 1,205
Posted By james hadfield
We've got excellent differential gene expression...

We've got excellent differential gene expression results with Illumina TruSeq stranded mRNA-seq and 10-20M single-end 50bp reads. We're seeing an issue with index number D703 but you can double check...
Forum: Illumina/Solexa 11-11-2015, 12:52 PM
Replies: 68
Views: 19,702
Posted By james hadfield
At Illumina's UK UGM the team discussed the low...

At Illumina's UK UGM the team discussed the low quality at the end of long-read MiSeq kits. They acknowleged there was an issue and that they were actively working on it. However they have no plans...
Forum: Sample Prep / Library Generation 11-11-2015, 12:44 PM
Replies: 2
Views: 1,301
Posted By james hadfield
Hi Alvaro and Martin, We have seen drop out of...

Hi Alvaro and Martin,
We have seen drop out of the column 3 (D703) index on multiple RNA-seq runs. We are now very confident that this is not a simple problem. If we run the same library on multiple...
Forum: Illumina/Solexa 08-15-2014, 04:56 AM
Replies: 58
Views: 34,032
Posted By james hadfield
Hi, I just wrote a new post on my blog,...

Hi, I just wrote a new post on my blog, http://core-genomics.blogspot.co.uk/2014/08/how-many-miseq-owners-are-using-bleach.html which describes the bleach protocol and how to get it.
Forum: Illumina/Solexa 08-15-2014, 04:54 AM
Replies: 10
Views: 6,994
Posted By james hadfield
How to get the bleach protocol

Hi, I just wrote a new post on my blog, http://core-genomics.blogspot.co.uk/2014/08/how-many-miseq-owners-are-using-bleach.html which describes the bleach protocol and how to get it.
Forum: Sample Prep / Library Generation 01-22-2014, 05:53 AM
Replies: 20
Views: 7,589
Posted By james hadfield
Forgot to direct you to this: Development of an...

Forgot to direct you to this: Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.

http://genomebiology.com/2013/14/12/R147/abstract
Forum: Sample Prep / Library Generation 01-22-2014, 05:51 AM
Replies: 2
Views: 2,000
Posted By james hadfield
You've over amplified the library with too many...

You've over amplified the library with too many PCR cycles and are seeing a "bubble' product. This is where the PCR reaction runs out of primer but annealing means the ends of 2 non-complementary...
Forum: Sample Prep / Library Generation 01-22-2014, 05:20 AM
Replies: 4
Views: 2,240
Posted By james hadfield
We've run 96 rapid-exomes but not done the direct...

We've run 96 rapid-exomes but not done the direct comparison to TruSeq etal. The on-target rates were good, duplication rate was a bit higher than we'd previously seen and at some level I'd say the...
Forum: Sample Prep / Library Generation 12-16-2013, 10:08 AM
Replies: 28
Views: 9,872
Posted By james hadfield
We're just about to test BioRads ddPCR method....

We're just about to test BioRads ddPCR method. Will let you know how it goes!
Forum: Sample Prep / Library Generation 12-16-2013, 10:00 AM
Replies: 20
Views: 7,589
Posted By james hadfield
We've one group making use of ChIP-exo the peaks...

We've one group making use of ChIP-exo the peaks look beautiful, are base-pair accurate for binding analysis and probably mean we only need 25% of the read depth. However the protocol is definitley...
Forum: Illumina/Solexa 12-16-2013, 09:57 AM
Replies: 12
Views: 5,656
Posted By james hadfield
I thought I should report on the problem we were...

I thought I should report on the problem we were seeing with read 2 index failures.
It turns out that we were seeing the affect of acidification of NaOH on the instrument, this meant the first...
Forum: General 10-21-2013, 01:40 AM
Replies: 7
Views: 2,677
Posted By james hadfield
Hmm, I'm not sure as I deal very much with the...

Hmm, I'm not sure as I deal very much with the wetlab side. I know we use Clarity as part of our automated analaysis but I believe we get the data out and use an in-house built workflow manager to...
Forum: RNA Sequencing 10-21-2013, 01:35 AM
Replies: 4
Views: 1,860
Posted By james hadfield
Option 3 every time. You can multiplex up to...

Option 3 every time.

You can multiplex up to 96 samples in one pool on HiSeq. I always recommend pooling the whole experiment if possible, and if not consulting with a statistician beforehand to...
Forum: Illumina/Solexa 10-21-2013, 01:33 AM
Replies: 18
Views: 4,449
Posted By james hadfield
We found the bead normalisation was OK but...

We found the bead normalisation was OK but relying on it to achieve the correct cluster density was a wate of time. Most users want 1 good MiSeq run and the QT is too falky to guarantee this.
We...
Forum: Illumina/Solexa 10-21-2013, 01:30 AM
Replies: 2
Views: 1,871
Posted By james hadfield
ScottC's right. Good pipetting, large volumes...

ScottC's right. Good pipetting, large volumes (>5ul) and serial dilution till you get to the required concentration. Don't even try and use Nanodrop!

Hopefully Illumina et al will move to a bead...
Forum: Illumina/Solexa 10-21-2013, 01:27 AM
Replies: 2
Views: 2,413
Posted By james hadfield
The clustering favours small molecules so if you...

The clustering favours small molecules so if you ahvea distribution of fragment sizes you may well see an imbalance compared to a Bioanalyser trace for instance. I'd go for a slightly lower cluster...
Forum: Illumina/Solexa 10-10-2013, 08:13 AM
Replies: 4
Views: 2,417
Posted By james hadfield
Rapid exome

has anyone used the newest rapid exome capture kits from Ilumina. Our first run generated huge amounts of library pre-caprute and again in teh final PCR. I'm worried about over-amplifying and adding...
Forum: RNA Sequencing 10-10-2013, 08:10 AM
Replies: 9
Views: 8,782
Posted By james hadfield
I'd recommend SE50bp for most DGE experiments as...

I'd recommend SE50bp for most DGE experiments as they are cheap and plentiful. More sample replicates is better than more reads to estimate within group variability better. We have done work on this...
Forum: Epigenetics 10-10-2013, 08:08 AM
Replies: 14
Views: 6,796
Posted By james hadfield
Why not look at Cambridge Epigenetix's new True...

Why not look at Cambridge Epigenetix's new True Methylome for oxidative bisulfite sequencing (oxBS-Seq)?
http://www.cambridge-epigenetix.com/en_US/products/1/truemethyl-6
Forum: Illumina/Solexa 10-10-2013, 08:04 AM
Replies: 12
Views: 5,656
Posted By james hadfield
Dual-index barcode read failures

Hi All,
We've seen a lot of read 2 failures on our HiSeq and on externally supplied data. The quality of the second index is terrible and we lose most of the reads as they cannot be...
Forum: General 10-10-2013, 07:53 AM
Replies: 7
Views: 2,677
Posted By james hadfield
We have now been using Clarity for six months or...

We have now been using Clarity for six months or so, three-four months for everything NGS in the lab and I'd say its been a success. Now we have everyone in the lab used to using it and users...
Forum: Sample Prep / Library Generation 05-10-2013, 12:46 PM
Replies: 16
Views: 29,679
Posted By james hadfield
Hmm, I'm not sure about the amplification primers...

Hmm, I'm not sure about the amplification primers having anything special on them. We use standard desalted oligos from Sigm for our amplification of Fluidigm libraries and we have run up to 1536...
Forum: Sample Prep / Library Generation 05-09-2013, 10:33 PM
Replies: 16
Views: 29,679
Posted By james hadfield
I'd seconddocbio's request for 384 barcodes ad...

I'd seconddocbio's request for 384 barcodes ad not just for Nextera XT. ChIP-seq and RNA-seq can often require more than 96 samples and we try to make a superpool and spread it across the number of...
Forum: Illumina/Solexa 05-09-2013, 10:31 PM
Replies: 7
Views: 2,312
Posted By james hadfield
We have a Tecan Evo but are mainly using it for...

We have a Tecan Evo but are mainly using it for qPCR setup and normalisations.
Showing results 1 to 25 of 221

 


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