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Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting., (© 2015 Salipante et al.; Published by Cold Spring Harbor Laboratory Press.)

Exome sequencing studies of autism spectrum disorders (ASDs) have identified many de novo mutations but few recurrently disrupted genes. We therefore developed a modified molecular inversion probe method enabling ultra-low-cost candidate gene resequencing in very large cohorts. To demonstrate the power of this approach, we captured and sequenced 44 candidate genes in 2446 ASD probands. We discovered 27 de novo events in 16 genes, 59% of which are predicted to truncate proteins or disrupt splicing. We estimate that recurrent disruptive mutations in six genes-CHD8, DYRK1A, GRIN2B, TBR1, PTEN, and TBL1XR1-may contribute to 1% of sporadic ASDs. Our data support associations between specific genes and reciprocal subphenotypes (CHD8-macrocephaly and DYRK1A-microcephaly) and replicate the importance of a β-catenin-chromatin-remodeling network to ASD etiology.

The relatively short read lengths associated with the most cost-effective DNA sequencing technologies have limited their use in de novo genome assembly, structural variation detection, and haplotype-resolved genome sequencing. Consequently, there is a strong need for methods that capture various scales of contiguity information at a throughput commensurate with the current scale of massively parallel sequencing. We propose in situ library construction and optical sequencing on the flow cells of currently available massively parallel sequencing platforms as an efficient means of capturing both contiguity information and primary sequence with a single technology. In this proof-of-concept study, we demonstrate basic feasibility by generating >30,000 Escherichia coli paired-end reads separated by 1, 2, or 3 kb using in situ library construction on standard Illumina flow cells. We also show that it is possible to stretch single molecules ranging from 3 to 8 kb on the surface of a flow cell before in situ library construction, thereby enabling the production of clusters whose physical relationship to one another on the flow cell is related to genomic distance.

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 © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We demonstrate the successful application of exome sequencing to discover a gene for an autosomal dominant disorder, Kabuki syndrome (OMIM%147920). We subjected the exomes of ten unrelated probands to massively parallel sequencing. After filtering against existing SNP databases, there was no compelling candidate gene containing previously unknown variants in all affected individuals. Less stringent filtering criteria allowed for the presence of modest genetic heterogeneity or missing data but also identified multiple candidate genes. However, genotypic and phenotypic stratification highlighted MLL2, which encodes a Trithorax-group histone methyltransferase: seven probands had newly identified nonsense or frameshift mutations in this gene. Follow-up Sanger sequencing detected MLL2 mutations in two of the three remaining individuals with Kabuki syndrome (cases) and in 26 of 43 additional cases. In families where parental DNA was available, the mutation was confirmed to be de novo (n = 12) or transmitted (n = 2) in concordance with phenotype. Our results strongly suggest that mutations in MLL2 are a major cause of Kabuki syndrome.

We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.

We demonstrate the first successful application of exome sequencing to discover the gene for a rare mendelian disorder of unknown cause, Miller syndrome (MIM%263750). For four affected individuals in three independent kindreds, we captured and sequenced coding regions to a mean coverage of 40x and sufficient depth to call variants at approximately 97% of each targeted exome. Filtering against public SNP databases and eight HapMap exomes for genes with two previously unknown variants in each of the four individuals identified a single candidate gene, DHODH, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway. Sanger sequencing confirmed the presence of DHODH mutations in three additional families with Miller syndrome. Exome sequencing of a small number of unrelated affected individuals is a powerful, efficient strategy for identifying the genes underlying rare mendelian disorders and will likely transform the genetic analysis of monogenic traits.

We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biology.

Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.