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The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian enhancers at single-nucleotide resolution through a massively parallel reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2-3% divergence from the wild-type sequence. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing the transcribed tags. Linear regression analysis yielded highly reproducible estimates of the effect of every possible single-nucleotide change on enhancer activity. The functional consequence of most mutations was modest, with ∼22% affecting activity by >1.2-fold and ∼3% by >2-fold. Several, but not all, positions with higher effects showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.

The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption-both intentionally and through widespread cross-contamination-and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes. [ABSTRACT FROM AUTHOR]

Recurrent deletions have been associated with numerous diseases and genomic disorders. Few, however, have been resolved at the molecular level because their breakpoints often occur in highly copy-number-polymorphic duplicated sequences. We present an approach that uses a combination of somatic cell hybrids, array comparative genomic hybridization, and the specificity of next-generation sequencing to determine breakpoints that occur within segmental duplications. Applying our technique to the 17q21.31 microdeletion syndrome, we used genome sequencing to determine copy-number-variant breakpoints in three deletion-bearing individuals with molecular resolution. For two cases, we observed breakpoints consistent with nonallelic homologous recombination involving only H2 chromosomal haplotypes, as expected. Molecular resolution revealed that the breakpoints occurred at different locations within a 145 kbp segment of >99% identity and disrupt KANSL1 (previously known as KANSL1). In the remaining case, we found that unequal crossover occurred interchromosomally between the H1 and H2 haplotypes and that this event was mediated by a homologous sequence that was once again missing from the human reference. Interestingly, the breakpoints mapped preferentially to gaps in the current reference genome assembly, which we resolved in this study. Our method provides a strategy for the identification of breakpoints within complex regions of the genome harboring high-identity and copy-number-polymorphic segmental duplication. The approach should become particularly useful as high-quality alternate reference sequences become available and genome sequencing of individuals'' DNA becomes more routine. [Copyright &y& Elsevier]