ASHG 2010, Post 3

Made it to the exome sequencing session this morning, and caught the end of the cancer sequencing project.  Some interesting points

• For pure tumor, 30x is fine, but for heterogeneous samples/samples with ploidy!=2, much more sequencing is needed.
• 150x for their exome samples was typical.
• Exome sequencing is now their hypothesis generating step
• With sequencing of a large number of carcinoma and multiple myloma samples, they found a number of mutations that would be unlikely to exist by chance, including about 10 that were not in their list of 6000 target cancer genes

In an iPS talk, they used a slightly more complicated exon capture:

• Padlock method, designed >300,000 probes, 600,000 synthesized.
• complemented with SureSelect
• NS/Syn ratio: typically .82:1

Jay Shendure on exome sequencing

• His group (with UW) has found 3 (2 novel) Mendelian 
• Total: about 10 found so far
• Working on exome sequencing in Autism
• In Autism
• Simplifying assumptions
• some % highly penetrant
• some % de novo
• more focused than looking for de novo CNV
• Paradigm: trio-based exome sequencing
• Very high SNR! by chance, expecting .59 mutations within coding regions--how to find this in the noise?
• So far: 20 trios (60 exomes) -- data from sporadic autism
• de novo SNV analysis pipeline: "Haystack"
1. look at bases called in all 3
2. ID Mendelian errors w/ discordant proband
3. Filter against >1000 other exomes (to eliminate false positive)
4. Annotate (SeattleSeq)
5. Manual review & Sanger confirmation
• Single trio ex
• 268 candidate den novo (mendelian errors0
• other exome screening -> 214
• manual review -> 18
• manual review -> 2 confirmed denovo events
• Not significant so far--need more numbers--but good trend
• Identified
• GRIN2B
• cause MR, epilepsy (Nature (genetics?) Nov 3)
• FOXP1
• related to FOXP2
• SCN1A, LAMC3: following up on these
• Parent of origin analysis
• molecular haplotyping by long range PCR & sequencing
• phase and determine parent of origin of each den novo point mutation
• So far: 7 from father, 2 from mother
• Model for potentially relevant for identifying large-effect non-coding mutations (when whole genome is cost-effective)
• NHLBI Exome Sequencing Project (ESP)
• Goal: 7000 exomes over 3 years
• Private coding variants in 1000 exomes
• several hundred per exome (combined Eurpoean American and African)
• Number of genes consistent with a domninant model
• dominant: roughly 100 +- 28 (1000 exomes filter), ~400 (1% allele freq)
• recessive: 2 +- 2 (1000 exomes filter), 1% allele freq (~35)
• Current/Future
• 50 nanogram exomes: fragmented with transposase (Andrew Adey)
• Using Nimblegen EZ Exome (2nd generation works well)
• Working toward multiplexing exomes on hiseq
• Currently targeting only 8x/exome? (might have misread/misheard this)

Goncalo Abecasis on draft sequencing of 1000 Genomes in Sardinia