Joshua N. Burton

Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions

Genomes assembled de novo from short reads are highly fragmented relative to the finished chromosomes of Homo sapiens and key model organisms generated by the Human Genome Project. To address this problem, we need scalable, cost-effective methods to obtain assemblies with chromosome-scale contiguity. Here we show that genome-wide chromatin interaction data sets, such as those generated by Hi-C, are a rich source of long-range information for assigning, ordering and orienting genomic sequences to chromosomes, including across centromeres. To exploit this finding, we developed an algorithm that uses Hi-C data for ultra-long-range scaffolding of de novo genome assemblies. We demonstrate the approach by combining shotgun fragment and short jump mate-pair sequences with Hi-C data to generate chromosome-scale de novo assemblies of the human, mouse and Drosophila genomes, achieving—for the human genome—98% accuracy in assigning scaffolds to chromosome groups and 99% accuracy in ordering and orienting scaffolds within chromosome groups. Hi-C data can also be used to validate chromosomal translocations in cancer genomes.

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

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.

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Species-level deconvolution of metagenome assemblies with Hi-C-based contact probability maps.

Microbial communities consist of mixed populations of organisms, including unknown species in unknown abundances. These communities are often studied through metagenomic shotgun sequencing, but standard library construction methods remove long-range contiguity information; thus, shotgun sequencing and de novo assembly of a metagenome typically yield a collection of contigs that cannot readily be grouped by species. Methods for generating chromatin-level contact probability maps, e.g., as generated by the Hi-C method, provide a signal of contiguity that is completely intracellular and contains both intrachromosomal and interchromosomal information. Here, we demonstrate how this signal can be exploited to reconstruct the individual genomes of microbial species present within a mixed sample. We apply this approach to two synthetic metagenome samples, successfully clustering the genome content of fungal, bacterial, and archaeal species with more than 99% agreement with published reference genomes. We also show that the Hi-C signal can secondarily be used to create scaffolded genome assemblies of individual eukaryotic species present within the microbial community, with higher levels of contiguity than some of the species’ published reference genomes.

A Year of Infection in the Intensive Care Unit: Prospective Whole Genome Sequencing of Bacterial Clinical Isolates Reveals Cryptic Transmissions and Novel Microbiota.

Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital’s intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care.

Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘Digital Deletion Detection’ (3D) that allows for high‐resolution analysis of rare deletions occurring at frequencies as low as 1 × 10−8. 3D is a three‐step process that includes targeted enrichment for deletion‐bearing molecules, single‐molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age‐related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre‐existing mutations is the primary factor contributing to age‐related accumulation of mtDNA deletions.

Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries

Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.

Accurate identification of centromere locations in yeast genomes using Hi-C

Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations. We describe a method, Centurion, that jointly infers the locations of all centromeres in a single genome from Hi-C data by exploiting the centromeres’ tendency to cluster in three-dimensional space. We first demonstrate the accuracy of Centurion in identifying known centromere locations from high coverage Hi-C data of budding yeast and a human malaria parasite. We then use Centurion to infer centromere locations in 14 yeast species. Across all microbes that we consider, Centurion predicts 89% of centromeres within 5 kb of their known locations. We also demonstrate the robustness of the approach in datasets with low sequencing depth. Finally, we predict centromere coordinates for six yeast species that currently lack centromere annotations. These results show that Centurion can be used for centromere identification for diverse species of yeast and possibly other microorganisms.

Single-molecule sequencing and conformational capture enable de novo mammalian reference genomes

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most contiguous de novo mammalian assembly to date, with chromosome-length scaffolds and only 663 gaps. Our assembly represents a >250-fold improvement in contiguity compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, supporting the most complete repeat family and immune gene complex representation ever produced for a ruminant species.

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi‐C to the assembly of genomes from mixed populations. Here, we show the methods application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de‐convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi‐C‐based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright