Technology

Several diverse platforms are involved in generating all facets of genomic data for the TCGA for each cancer type. However, the Broad Institute has specialized and is mainly responsible for two technologies in specific capacities.

The established platforms used at the Broad Institute to generate TCGA data are the Affymetrix SNP 6.0 Array for genotyping and copy number inference and the Illumina Genome Analyzer IIx system for paired-end sequencing data.

While we are processing many TCGA sample with the above platforms we are also piloting many new and cutting edge technologies such as single cell sequencing, jumping libraries, and the Illumina Cyto12 SNP array platform.

Affymetrix SNP 6.0

The SNP 6.0 array is used to genotype samples from the numerous SNP markers the chip can probe for. Copy-number can also be inferred from the array probes simultaneously. The array was designed in conjunction with the Broad Institute and the descriptive publication of the technology is below.

All technical documentation regarding the technology and further description can be found at the official Affymetrix product page here.

References

McCarroll SA, et al. “Integrated detection and population-genetic analysis of SNPs and copy number variation.” Nature Genetics (2008) 40(10):1166-1175. [PMID: 18776908]

Illumina Genome Analyzer (GA) IIx

The GAIIx platform is used to generate paired-end sequence data. As a next-generation sequencing machine it can process samples from whole genome DNA as well as more targeted captures like poly-T pulldown (RNA-Seq) or hybrid capture (exome sequencing). A more comprehensive description as well as manufacturer protocols can be found at the official Illumina product page here.

References

Official Illumina GA IIx Product Page

Single Cell Sequencing

segseq

Comparison of Copy Number profiles from Traditional Bulk Sequencing versus Single-Cell Sequencing.

Biological and clinical significance of tumor heterogeneity remain poorly understood despite major advances in cancer genome analysis techniques, especially next generation sequencing technologies. Therefore we have initiated a technology development project to enable genome and transcriptome sequencing sequencing from single cells. To this end, we have adapted the whole genome amplification (WGA) protocol to single cells based on Multiple Displacement Amplification (MDA) technology. So far, we have sequenced two single cell genome libraries from FACS sorted HeLa cells. Each library was sequenced to a depth of ~35 million paired-end 36 bp sequences using the GA IIx sequencer. 98% of sequences align to hg19 with a less than 2% duplication rate. Mean coverage of the genome for both libraries was 0.9X. We have also developed a multiplex quantitative NanostringTM assay comprised of 329 randomly chosen genomic target sequences, which allows rapid, reproducible, and cost-effective measurement of DNA copy number profiles from single cell libraries relative to the reference libraries from bulk genomic DNA.

References

Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, Cook K, Stepansky A, Levy D, Esposito D, Muthuswamy L, Krasnitz A, McCombie WR, Hicks J, Wigler M. “Tumour Evolution Inferred by Single-cell Sequencing.” Nature (2011) 474(7341):90-4. [PMID: 21399628] [Free: NA]

Chiang DY, Getz G, Jaffe DB, O’Kelly MJ, Zhao X, Carter SL, Russ C, Nusbaum C, Meyerson M, Lander ES. “High-resolution mapping of copy-number alterations with massively parallel sequencing.” Nature Methods (2009) 6(1):99-103. [PMID: 19043412] [Free: PubMed]

Official Qiagen REPLI-g Kit for WGA

Jumping Libraries

Identification of gross chromosomal alterations and de novo genome assembly may be possible in low pass sequencing of tumors through the use of DNA jumping libraries. The Broad Sequencing Platform (BSP) has recently developed a protocol to generate jumping libraries for Illumina’s next generation sequencing platform. This protocol is currently being tested to determine if jumping libraries can be used to detect genomic rearrangements using cancer cell line K562.

References

N/A

Illumina Cyto12 SNP Array

IGV Comparison of Copy Number derived from SNP6 versus Cyto12 platforms.

We have completed initial experiments to determine whether a low cost ~300K Cyto12 Array from Illumina is sufficient for ABSOLUTE analysis. In conjunction with greatly reduced amounts of sample DNA (15 ng), Cyto12 arrays have successfully been used to produce segmented copy number data. Experiments to determine the reproducibility of these arrays are currently in progress.

References

Official Illumina Cyto12 Product Page

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