NGS: understanding cancer genomes

http://www.nature.com/nrg/journal/v11/n10/pdf/nrg2841.pdf

impressive review... read many times please!

Combining homozygosity mapping with exome capture: SDCCAG8 and retinal-renal ciliopathy

http://www.nature.com/ng/journal/v42/n10/full/ng.662.html

Combining homozygosity mapping with exome capture

The finding that most known NPHP-RC genes caused the disorder only in a small number of cases (<1%)⁹necessitated the ability to map and identify disease genes in single families. We therefore developed a strategy that combines homozygosity mapping in single families¹³ with exon capture and consecutive massively parallel sequencing¹⁴. Using the NimbleGen 385K platform, we designed a ciliopathy candidate exon capture array, which contains oligonucleotides that interrogate ~13,000 exons from the 'UCSC Gene' annotation (see URLs) of 828 NPHP-RC candidate genes. Candidate genes were derived from ciliopathy animal models, from the photoreceptor sensory cilia proteome¹⁵ and from other candidate sources¹⁶(Supplementary Tables 1–3).

Because exon capture with subsequent massively parallel sequencing yields too many variants from normal reference sequence (VRSs) to make a safe call regarding the disease-causing mutation¹⁴, we devised a strategy of a priori reduction of VRSs (Supplementary Table 1). These a priori restriction criteria consisted of: (i) capturing only ~13,000 ciliopathy candidate exons instead of all ~180,000 exons from the collaborative consensus coding sequence (CCDS) project (~15-fold reduction; Supplementary Table 1); (ii) evaluating coding SNPs, splice variants and indels only (as other variants will be difficult to interpret); (iii) removing VRSs from a database of innocuous SNPs (dbSNP130; 2.3-fold reduction); (iv) evaluating only within the mapped homozygous candidate region of an individual or family (~20-fold reduction); and (v) preferentially evaluating truncating mutations (~4-fold reduction). This approach allowed us to reduce the number of VRSs by an average of ~2,760-fold and led to the identification of the disease-causing gene in 3 out of 5 attempts (Supplementary Table 1). We discovered homozygous mutations in the known NPHP-RC genes AHI1 (family A2045) and INVS (family A128; Supplementary Table 1). More importantly, we discovered a homozygous mutation in SDCCAG8 as a new cause of NPHP-RC (Supplementary Table 1).

NGS Statistics: GRIN2A and melanoma by Yardena Samuels

http://www.nature.com/ng/journal/v43/n5/pdf/ng.810.pdf

We conducted an exome resequencing of 14 matched normal and metastatic tumor DNAs from untreated individuals with melanoma. We enriched exonic sequences using Agilent's SureSelect technology for targeted exon capture⁶, targeting 37 Mb of sequence from exons and their flanking regions in ~20,000 genes. We performed sequencing with the Illumina GAII platform and aligned the reads using ELAND (Illumina, Inc.) followed by cross_match (see URLs) to the reference human genome (build 36.1). On average, we generated 12 Gb of sequence per sample to a mean depth of 180× or greater to achieve exome builds with at least 90% of the exons covered by high quality genotype calls. To eliminate common germline mutations, we removed any potential somatic mutation that was observed in dbSNP130 or in the 1000 Genomes Project data. To determine which of these alterations were somatic (that is, tumor-specific), we compared these data to the matched normal tissue. From these putative alterations, we identified 5,161 potential somatic mutations in 3,568 different genes in the 14 samples sequenced.