>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
* 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…]