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Saturday, February 27, 2010

AGBT 2010 - Keynote: Henry Erlich - Roche Molecular Systems

Applications of Next Generation Sequencing: HLA Typing With the GSFLX System

High Throughput HLA typing
* the allelic diversity is enormous
* Focussing on HLA class I and II genes (germ-line)

Challengeing because it's the most polymorphic region in the genome
* HLA-B has well over 1000 alleles
* only 68 different serological types can be distinguished
* 3,529 genes at 12 loci as of April 2009
* chromosome 6
* Can't be typed using existing conventional techniques [I assume in high throughput]
* DR-DQ region - involved in type I diabetes
[Much detail here, which I can't get down fast enough with any hope at accuracy.]

Polymorphism is highly localized.
* virtually all of the polymorphic amino acid residues are localized to a groove.
* most allelic differences are protein coding.
* critical to distinguish known alleles

* eg HLA-A * 24020101
* only the first 4 numbers are the ones that distinguish the protein.

Survival curve for bone marrow transplant
* even with 8/8 allele matches, there are WAY more things that need to be matched - and so you need the best possible match.
* a single coding mismatch can cause graph vs host disease.
* Bone Marrow matching requires high precision

[List of disease applications - 22 different diseases including Narcolepsy, cancers, drug allergic reactions..]

GWAS in Type 1 diabetes.
* identified disease related genes - HLA SNPs are significant
* Dr-DQ haplotypes are associated strongly with Odds ratio for diabetes
* looking at genomic risk factors increase up to 40x

[something about a particular combination of DR-DQ giving VERY high risk, and consequently is never seen in humans...]

* Dot blots... evolved into Probe Array Typing System.
* Even if you have hundreds of probes, you still have "HLA Genotye Ambiguity"
* "Fail to distinguish alleles" without NGS (with or without phasing..)

[Explanation of how 454 works - protocol]

* amplify exons with MID primers/emPCR/sequence

Benefits of clonal sequencing
* set phase to reduce ambiguity
* allow amplification and sequencing of multiple members of multi-gene family with generic primers
* allow sorting /separation of co-amplified sequences from target sequence (signal)

Parallel clonal sequencing of 8 loci x 24 samples

[More protocol... ]

Graph of read length : around 250bp

Connexio Assignment of DRB1 Genotype
* image reassuring to a HLA researcher.
* like the interface (plug for the company)
* aligns sequence, consensus sequence, does genotype assignment
* [Must admit, the information on this interface is rather mysterious to me...]
* [Several more slides of Connexio data and immunology types that mean nothing to me.]
* get a genotype report...


Testing on SCIDS patient
* patients are potentially chimeric
* look for presence of non-transmitted maternal allele
* can find stuff in "fail layer" because software assumes only two alleles possible.

[Wow... I know I don't know much immunology, but I'm not getting much out of this. This is a lot of software for immunologists, and I really don't understand the terminology, making it challenging to get coherent notes.]

Takes about 4 days - [says 5-7 on the slide]
* amplicon prep
* emulsion
* DNA bead process
* loading wells
* sequencing on GSLFX
* Data analysis

[Missed slide on how much data they were getting - 1M reads?]

Multiplex - 500 samples in one run
* Got good results [not copying down seemingly random DRB numbers...]



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