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Forum: Illumina/Solexa 03-07-2011, 10:16 AM
Replies: 1
Views: 4,345
Posted By Bruce E
Fastqc sequence duplication levels

Our informatics group recently started to use fastqc. I am seeing high (70%) sequence duplication for TruSeq mRNA-Seq libraries. These are from a single lane of a 51 bp read on Illumina's HiSeq...
Forum: Illumina/Solexa 02-07-2011, 01:02 PM
Replies: 1
Views: 3,081
Posted By Bruce E
Indexed Sure Select Capture

We want to try making PEIn Illumina libraries and then mixing them followed by Sure Select whole exon cap and PEIn sequencing. Has anyone tried this and what did you use for blocking during capture?
Forum: Illumina/Solexa 01-10-2011, 11:33 AM
Replies: 2
Views: 8,736
Posted By Bruce E
Index PCR

Your original thought is incorrect. In all PCR the pcr primers become part of your product. The only difference with index pcr is that you add an index sequnce to the 5' end of your pcr primer.
Forum: Illumina/Solexa 07-13-2010, 09:09 AM
Replies: 3
Views: 4,647
Posted By Bruce E
The A overhang is also needed so the adapter...

The A overhang is also needed so the adapter ligates in the correct orientation. They refer to these as adapters but it is actually one Y adapter so you need it to ligate in the correct orientation...
Forum: Illumina/Solexa 06-09-2010, 06:37 AM
Replies: 17
Views: 9,845
Posted By Bruce E
mRNA seq

We have been running RNA-seq with out a PhiX for over a year and it works fine. Giving up a lane for a standard is too expensive. The only reason you need a PhiX besides QC is if you are doing an...
Forum: Illumina/Solexa 04-14-2010, 09:43 AM
Replies: 1
Views: 2,022
Posted By Bruce E
Primer Dimers in PEIn libraries

We are preparing PEIn libraries starting with 200 ng of concantenated Raindance products. We used 10X less adapter mix when ligating the adapters to adjust for the lower starting DNA concentration....
Forum: Sample Prep / Library Generation 03-11-2010, 08:43 AM
Replies: 6
Views: 7,466
Posted By Bruce E
Illumina Mate Pair Prep

Did you ever find out what these were. My Mate pair kit is on backorder and I am trying to by reagents separately. I am also trying to which DNA Exonuclease they use.
Forum: Illumina/Solexa 02-24-2010, 01:50 PM
Replies: 2
Views: 3,127
Posted By Bruce E
Whole Exome Capture

We just finished a GAIIx 76 X 2 PE run of libraries prepared with Agilents Whole exome Sure Select kit. We analysed them using the Illumina Pipeline and aligned them to the human genome. The overall...
Forum: RNA Sequencing 07-28-2009, 03:37 PM
Replies: 1
Views: 5,579
Posted By Bruce E
Small RNA sequencing on Illumina

2 questions. What are people using to isolate total RNA for the small RNA prep and has anyone found a way to multiplex small RNA samples?
Forum: Illumina/Solexa 05-28-2009, 12:09 PM
Replies: 7
Views: 4,604
Posted By Bruce E
We are starting a comparison too. I will post our...

We are starting a comparison too. I will post our results. I was wondering if anyone has tried SCS 2.4 yet and if they would care to share their experiences.
Thanks
Forum: RNA Sequencing 05-12-2009, 01:53 PM
Replies: 1
Views: 2,953
Posted By Bruce E
Error rate of 51 cycle mRNA Seq

We have been running mRNAseq on an Illumina GAll. At cycle 36 we are seeing a spike in our error rate. Has anyone else observed this? We do not see it when we run genomic dna or in our standard.
Forum: Illumina/Solexa 03-05-2009, 06:31 AM
Replies: 6
Views: 2,157
Posted By Bruce E
We have sequenced human mito DNA on the Illumina...

We have sequenced human mito DNA on the Illumina and get very good results.
Forum: Illumina/Solexa 12-08-2008, 06:47 AM
Replies: 0
Views: 1,697
Posted By Bruce E
Question ChIP Seq

Does anyone have a good feel for what size gel slice should be used in preparing Illumina ChIP Seq libraries? I have been using a product called gene catcher and it cuts out a slice that is 1 mm wide...
Forum: General 11-24-2008, 08:35 AM
Replies: 0
Views: 2,556
Posted By Bruce E
Fragmentation

I am considering getting a Covaris E210 (96 well capable) for fragmentation for sequencing on the Illumina & Applied Bio Solid sequencing systems. Does antone have experience they could share using...
Forum: Sample Prep / Library Generation 03-14-2008, 01:12 PM
Replies: 29
Views: 26,284
Posted By Bruce E
Nebulization, other shearing methods

I cannot get dna to fragment to 200bp. I am using nebulization protocol from Illumina. I have tried 35psi 6 minutes, 35psi 8 min, 35 psi 10 min, 37psi 6 min, & 40psi 6 min. I am trying this on dna...
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