Ernest T. Lam

Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly.

We describe genome mapping on nanochannel arrays. In this approach, specific sequence motifs in single DNA molecules are fluorescently labeled, and the DNA molecules are uniformly stretched in thousands of silicon channels on a nanofluidic device. Fluorescence imaging allows the construction of maps of the physical distances between occurrences of the sequence motifs. We demonstrate the analysis, individually and as mixtures, of 95 bacterial artificial chromosome (BAC) clones that cover the 4.7-Mb human major histocompatibility complex region. We obtain accurate, haplotype-resolved, sequence motif maps hundreds of kilobases in length, resulting in a median coverage of 114× for the BACs. The final sequence motif map assembly contains three contigs. With an average distance of 9 kb between labels, we detect 22 haplotype differences. We also use the sequence motif maps to provide scaffolds for de novo assembly of sequencing data. Nanochannel genome mapping should facilitate de novo assembly of sequencing reads from complex regions in diploid organisms, haplotype and structural variation analysis and comparative genomics.

Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity

Mutations in genes on the nucleotide excision repair pathway are associated with diseases, such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that involve skin cancer and developmental and neurological symptoms. These mutations cause the defective repair of damaged DNA and increased transcription arrest but, except for skin cancer, the links between repair and disease have not been obvious. Widely different clinical syndromes seem to result from mutations in the same gene, even when the mutations result in complete loss of function. The mapping of mutations in recently solved protein structures has begun to clarify the links between the molecular defects and phenotypes, but the identification of additional sources of clinical variability is still necessary.

Temporal Dissection of Tumorigenesis in Primary Cancers

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.

A hybrid approach for de novo human genome sequence assembly and phasing

Despite tremendous progress in genome sequencing, the basic goal of producing a phased (haplotype-resolved) genome sequence with end-to-end contiguity for each chromosome at reasonable cost and effort is still unrealized. In this study, we describe an approach to performing de novo genome assembly and experimental phasing by integrating the data from Illumina short-read sequencing, 10X Genomics linked-read sequencing, and BioNano Genomics genome mapping to yield a high-quality, phased, de novo assembled human genome.

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.

Sequencing of TNFAIP3 and association of variants with multiple autoimmune diseases

The TNFAIP3 locus at 6q23, encoding A20, has been associated with multiple autoimmune diseases (AIDs). In this study, we sequence the coding portions of the gene to identify contributing causal polymorphisms that may explain some of the observed associations. A collection of 123 individuals from the Multiple Autoimmune Disease Genetics Consortium (MADGC) collection, each with multiple AIDs (mean=2.2 confirmed diagnoses), and 397 unrelated healthy controls were used for initial sequencing. A total of 32 polymorphisms were identified in the sequencing experiments, including 16 novel and 11 coding variants. Association testing in the entire MADGC collection (1,008 Caucasians with one or more AIDs and 770 unaffected family controls) revealed association of a novel intronic insertion–deletion polymorphism with rheumatoid arthritis (RA) (odds ratio (OR)=2.48, P=0.041). Genotyping of the most common coding polymorphism, rs2230926, in the MADGC collection and additional control individuals revealed a significant association with Sjögrens syndrome (OR=3.38, P=0.038), Crohns disease (OR=2.25, P=0.041), psoriasis (OR=0.037, P=0.036) and RA (OR=1.9, P=0.025). Finally, haplotype and additional testing of polymorphisms revealed that cases were enriched for 5′ and 3′ untranslated region variants (one-sided P-value=0.04), but not specifically for common (>2% minor allele frequency), rare, exonic, intronic, non-synonymous or synonymous variants.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.

Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis.

Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

Tools and pipelines for BioNano data: molecule assembly pipeline and FASTA super scaffolding tool

BackgroundGenome assembly remains an unsolved problem. Assembly projects face a range of hurdles that confound assembly. Thus a variety of tools and approaches are needed to improve draft genomes.ResultsWe used a custom assembly workflow to optimize consensus genome map assembly, resulting in an assembly equal to the estimated length of the Tribolium castaneum genome and with an N50 of more than 1 Mb. We used this map for super scaffolding the T. castaneum sequence assembly, more than tripling its N50 with the program Stitch.ConclusionsIn this article we present software that leverages consensus genome maps assembled from extremely long single molecule maps to increase the contiguity of sequence assemblies. We report the results of applying these tools to validate and improve a 7x Sanger draft of the T. castaneum genome.