Illumina/Solexa sequencing of human exons enriched with Nimblegen arrays

[ECO]

To continue this topic, as it's been pretty popular...there was just another paper released by another group at Cold Spring Harbor using Nimblegen arrays in combination with Solexa sequencing.

Link to Nature is here:
http://www.nature.com/ng/journal/v39...g.2007.42.html

Some interesting highlights below, I'll try to expand on the paper tomorrow...it's getting late for me!

1. They show what we've already seen from the Baylor/Emory groups (discussed over in our 454 forum), that it's possible to capture a majority of human exons on 6-7 Nimblegen arrays with ~350k features each. Seems to be the same probe design, 60-mers tiled every 20bp.

2. Some data that suggests WGA samples miss quite a bit...~60% of exons represented with WGA, and ~98% without. Also WGA biases slightly against AT rich exons.

3. Showed that shorter captured samples (150-200, the range Illumina recommends...) work much better than capturing 500-600 base fragments. This is not surprising as Illumina relies on fragment length for efficient bridging and cluster generation. Seems they tried the same protocol that the 454/Nimblegen groups used.

4. A remarkable lack of commentary on sequencing accuracy, with only one comment stating that they detected 60% of HapMap SNP's expected in this CEPH individual.

They show some actual read data in the Supplementary Info from five SNP loci, which in reality is quite disappointing from an accuracy standpoint. There are at least 3 random differences from consensus in some of the reads that they fail to address, and in the same breath claim they are calling heterozygous SNPs simply because there are differences seen at an rs locus. I don't have the paper in front of me now but there were quite a few low quality bases as well. Up to 17 bases out of 26 in one run were quite poor.

I also wonder why the authors, in the intro to the paper, describe Solexa sequencing as generating "35-50" bp reads, and then use 26bp read lengths throughout their study. Comes across like an industrial application note from Illumina/Nimblegen, rather than a Nature paper.

Anyway, very interesting paper, although not the best presentation of actual sequence data nor any biology. It reminds me of the early days of microarrays when all you had to do demonstrate a run on someone's array platform to get a Nature paper. In all actuality I'm just jealous that I don't get to work with this type of technology, yet.

[ivoire]

I have to clarify that WGA samples do not miss ~ 60% of exons. Read Okou et al (2007). they not only use WGA on all the samples (MDA method of repli g kit - Qiagen) but sequenced unique coding and non coding sequences. Not only did they get great basecall result but there is no reason to believe that WGA is particularly bias toward exons and not introns. for more information please read the paper by Pinard et al (BMC Genomics. 2006 Aug 23;7:216) and also BMC Biotechnol. 2005 Sep 16;5:24.

[ECO]

I have to clarify that WGA samples do not miss ~ 60% of exons. Read Okou et al (2007). they not only use WGA on all the samples (MDA method of repli g kit - Qiagen) but sequenced unique coding and non coding sequences. Not only did they get great basecall result but there is no reason to believe that WGA is particularly bias toward exons and not introns. for more information please read the paper by Pinard et al (BMC Genomics. 2006 Aug 23;7:216) and also BMC Biotechnol. 2005 Sep 16;5:24.

Hey ivoire, I think I was unclear with my wording...I meant that 60% WERE represented with WGA, versus 98% being represented without.

[sathish]

Difference between exon capture microarrays and gene expression microarrays

Hi Group,

I am new to next generation sequencing. I am wondering what is the difference between exon capture microarrays and gene expression microarrays?

I like to know the answers with respect transcript abundance and probe saturation.

Thanks a lot

[ECO]

Hi Group,

I am new to next generation sequencing. I am wondering what is the difference between exon capture microarrays and gene expression microarrays?

I like to know the answers with respect transcript abundance and probe saturation.

Thanks a lot

Hey sathish...gene expression microarrays are just that, for assaying gene expression! (but you knew that).

"Exon capture" or any type of enrichment array is basically an upstream sample prep method for genomic sequencing. It is one of a few methods for genome decomplexing, so that one can focus the next gen instrument on a specific region of interest instead of the entire genome. It doesn't just have to be exons that are captured, this was basically a proof of principle from Nimblegen, I personally think there are better uses than just exon sequencing.

Before array enrichment came on the scene, people do many specific PCRs to enrich for their regions of interest, then pool them prior to sequencing. This is totally workable but can be labor intensive for large regions or many patients.

Supposedly, Agilent is about to release a "solution phase array" which captures the regions of interest in solution rather than on solid phase, which should give improved kinetics of hybridization.

Hope that helps!

[sathish]

Microarays

Thanks for your reply.

However, I am wondering will there be a difference in exon or DNA capture. Can we use capture microarray to estimate the transcript abundance? Will capture arrays show difference between most abundant, intermediately abundant and low abundant transcripts?

Is there any change in array manufacture between expression arrays and capture arrays? Is there difference in probe saturation?

I think I am asking too many questions, basically I like to know technical difference between the two arrays.

Thanks again in advance for your answers.

Sathish

[2007lab]

I personally think there are better uses than just exon sequencing.


Supposedly, Agilent is about to release a "solution phase array" which captures the regions of interest in solution rather than on solid phase, which should give improved kinetics of hybridization.

Hi ECO, Just wondering what kind of uses do you forsee for the capture array?

Also do you have a link (news/ gossip/tech paper) to the Agilent solution array?

[colicoli]

News:

http://www.agilent.com/about/newsroom/presrel/2009/05feb-ca09006.html

and

http://www.opengenomics.com/Products/SureSelect_Target_Enrichment_System

Gossip:

http://www.genomeweb.com/arrays/despite-recent-deals-future-array-and-next-gen-sequencer-synergy-uncertain

Paper:

http://www.opengenomics.com/GnirkeReview

[FLXTech]

Seqcap service

Hi, do you know of any service providers for NimbleGen or Agilent's SureSelect seqcap technologies?

Thanks

[PNG]

sequence capture

Ambry Genetics:

http://www.ambrygen.com/Research_Support_Services/Research_Projects.asp

We developed our own sequence capture protocol for Illlumina libraries using Nimblegen arrays. We can also perform the standard protocol with the Nimblegen adapters if you have another sequencing platform. Or, we can sequence your samples here.

Please feel free to contact me for additional information.

Thanks,
Phillip

[email protected]