The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line

TL;DR

The paper reports a haplotype-resolved genome and epigenome of the aneuploid HeLa CCL-2 cancer cell line, using clone dilution pool sequencing to phase variants across full chromosome arms. It finds that HeLa is relatively stable in point variation, maps aneuploidy/LOH and structural rearrangements, and shows haplotype-specific activation of MYC by an integrated HPV-18 genome at chromosome 8q24.21.

Key findings

• HeLa CCL-2 carried 4.1 × 10^6 SNVs, with 90.2% overlapping 1000 Genomes Project variants, similar to control genomes (mean 4.2 × 10^6 SNVs; 87.7% overlap), suggesting no appreciably large accumulation of somatic SNVs relative to inherited variants.
• Copy-number analysis found 61% of the genome at baseline copy number three, only 3% at CN>4 or CN<2, and LOH encompassing 15.7% of the genome, including entire chromosome arms 5p, 6q, Xp, and Xq.
• The study validated 8,165 somatic mutations with an estimated sensitivity of 61.1%, giving a point mutational frequency of 2.16 per Mb, on the lower end of frequencies observed across cancer genomes.
• A highly rearranged HPV-18 integration at chromosome 8q24.21 was reconstructed; the integrated viral genome lacks a functional E2 and is associated with strong, haplotype-specific MYC activation ~500 kb upstream (mean expression ratio 95:1).

Methods

• Haplotype-resolved whole-genome sequencing of HeLa CCL-2 used shotgun sequencing to 88X non-duplicate coverage plus mate-pair, long-read, and clone dilution pool sequencing.
• Phasing used 288 fosmid clone dilution pools, yielding 518,293 non-overlapping clones with a median insert size of 33 kb and 6.3X physical coverage of the haploid reference; haplotype blocks had an N50 of 550 kb and scaffolds covered 1.96 Gb (90.3% of the non-LOH genome) with an N50 of 44.8 Mb.
• Copy number and LOH were mapped with HMM-based read-depth analysis; RNA-Seq and ENCODE datasets were phased against HeLa CCL-2 haplotype and copy-number maps to analyze haplotype-specific expression, chromatin, and transcription factor binding.

Limitations

• The paper states that normal tissue corresponding to HeLa is unavailable, so inherited and somatic variants cannot be fully distinguished; some protein-altering variants could not be excluded as rare inherited variants or passenger mutations.
• The paper states that without estimates for parameters such as the number of doublings during tumorigenesis, cells explanted, and passages in culture, the post-aneuploidy mutational burden cannot be rescaled to a rate per base per division.
• The paper states that further studies are required to resolve the functional relevance of candidate genes and to assess whether they are recurrently altered in HPV+ cervical carcinomas; it also notes that phasing of HeLa S3 ENCODE data relied on CCL-2 maps because the strains are nearly identical, though not identical.