Haplotype-resolved sequencing and structural analysis of the HeLa cancer genome. A. C. Adey, J. O. Kitzman, J. Burton, J. Shendure Genome Sciences Dept, University of Washington, Seattle, WA.

   We present the first haplotype-resolved sequence of a cancer genome. A PubMed search of HeLa returns over 68,000 hits. However, a comprehensive genomic analysis of the oldest and arguably most prevalent human cancer cell line has yet to be performed. We sequenced the HeLa genome to ~88X shotgun coverage in order to accurately identify variants in the context of extreme aneuploidy. Next, we constructed and sequenced nearly 300 fosmid clone pools, each containing ~2 percent representation of the genome, thereby providing islands of haplotype-specific coverage. In addition, we performed single chromosome flow-sorting and pooled sequencing in order to boost our ability to determine haplotype relationships amongst germline variants and somatic mutations across entire chromosomes, as well as to accurately catalog chromosomal rearrangements. Shotgun, fosmid clone pool, chromosome sorting, and 4-40 Kb mate-pair data were integrated to generate the first haplotype-resolved sequence of a cancer genome. The genome of HeLa may yield insights into the biological basis for its persistence and robust growth properties. Furthermore, we are using the haplotype information to estimate the timing and order of somatic events over the 60 year history of HeLa. Finally, it has been previously shown that a number of chromosomal fragments are present in populations of HeLa cells at varying frequencies. To further investigate this, we performed flow sorting and shotgun sequencing of single nuclei to characterize the content and frequencies of these abnormal chromosomes. The methods presented here may prove broadly useful for improving the comprehensiveness and resolution at which we characterize cancer genomes.

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