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July 7, 2019

Genome sequence of “Candidatus Microthrix parvicella” Bio17-1, a long-chain-fatty-acid-accumulating filamentous actinobacterium from a biological wastewater treatment plant.

Candidatus Microthrix bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: “Candidatus Microthrix parvicella” strain Bio17-1.


July 7, 2019

Cancer genomics: technology, discovery, and translation.

In recent years, the increasing awareness that somatic mutations and other genetic aberrations drive human malignancies has led us within reach of personalized cancer medicine (PCM). The implementation of PCM is based on the following premises: genetic aberrations exist in human malignancies; a subset of these aberrations drive oncogenesis and tumor biology; these aberrations are actionable (defined as having the potential to affect management recommendations based on diagnostic, prognostic, and/or predictive implications); and there are highly specific anticancer agents available that effectively modulate these targets. This article highlights the technology underlying cancer genomics and examines the early results of genome sequencing and the challenges met in the discovery of new genetic aberrations. Finally, drawing from experiences gained in a feasibility study of somatic mutation genotyping and targeted exome sequencing led by Princess Margaret Hospital-University Health Network and the Ontario Institute for Cancer Research, the processes, challenges, and issues involved in the translation of cancer genomics to the clinic are discussed.


July 7, 2019

Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011.

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May-July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.


July 7, 2019

Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory.

Recent methods have been developed to perform high-throughput sequencing of DNA by Single Molecule Sequencing (SMS). While Next-Generation sequencing methods may produce reads up to several hundred bases long, SMS sequencing produces reads up to tens of kilobases long. Existing alignment methods are either too inefficient for high-throughput datasets, or not sensitive enough to align SMS reads, which have a higher error rate than Next-Generation sequencing.We describe the method BLASR (Basic Local Alignment with Successive Refinement) for mapping Single Molecule Sequencing (SMS) reads that are thousands of bases long, with divergence between the read and genome dominated by insertion and deletion error. The method is benchmarked using both simulated reads and reads from a bacterial sequencing project. We also present a combinatorial model of sequencing error that motivates why our approach is effective.The results indicate that it is possible to map SMS reads with high accuracy and speed. Furthermore, the inferences made on the mapability of SMS reads using our combinatorial model of sequencing error are in agreement with the mapping accuracy demonstrated on simulated reads.


July 7, 2019

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype.

Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.


July 7, 2019

Next-generation sequencing and large genome assemblies.

The next-generation sequencing (NGS) revolution has drastically reduced time and cost requirements for sequencing of large genomes, and also qualitatively changed the problem of assembly. This article reviews the state of the art in de novo genome assembly, paying particular attention to mammalian-sized genomes. The strengths and weaknesses of the main sequencing platforms are highlighted, leading to a discussion of assembly and the new challenges associated with NGS data. Current approaches to assembly are outlined and the various software packages available are introduced and compared. The question of whether quality assemblies can be produced using short-read NGS data alone, or whether it must be combined with more expensive sequencing techniques, is considered. Prospects for future assemblers and tests of assembly performance are also discussed.


July 7, 2019

Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.

Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, ß-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic ß-catenin signalling in medulloblastoma.


July 7, 2019

An Inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an aggressive subtype of pediatric acute megakaryoblastic leukemia.

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.


July 7, 2019

Strobe sequence design for haplotype assembly.

Humans are diploid, carrying two copies of each chromosome, one from each parent. Separating the paternal and maternal chromosomes is an important component of genetic analyses such as determining genetic association, inferring evolutionary scenarios, computing recombination rates, and detecting cis-regulatory events. As the pair of chromosomes are mostly identical to each other, linking together of alleles at heterozygous sites is sufficient to phase, or separate the two chromosomes. In Haplotype Assembly, the linking is done by sequenced fragments that overlap two heterozygous sites. While there has been a lot of research on correcting errors to achieve accurate haplotypes via assembly, relatively little work has been done on designing sequencing experiments to get long haplotypes. Here, we describe the different design parameters that can be adjusted with next generation and upcoming sequencing technologies, and study the impact of design choice on the length of the haplotype.We show that a number of parameters influence haplotype length, with the most significant one being the advance length (distance between two fragments of a clone). Given technologies like strobe sequencing that allow for large variations in advance lengths, we design and implement a simulated annealing algorithm to sample a large space of distributions over advance-lengths. Extensive simulations on individual genomic sequences suggest that a non-trivial distribution over advance lengths results a 1-2 order of magnitude improvement in median haplotype length.Our results suggest that haplotyping of large, biologically important genomic regions is feasible with current technologies.


July 7, 2019

Direct detection and sequencing of damaged DNA bases.

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template – as a by-product of the sequencing method – through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.


July 7, 2019

Real-time sequencing.

This month’s Genome Watch describes the impact of next-generation sequencing on the ‘real-time’ analysis of pathogen genomes during outbreaks.


July 7, 2019

Computational solutions to large-scale data management and analysis.

Today we can generate hundreds of gigabases of DNA and RNA sequencing data in a week for less than US$5,000. The astonishing rate of data generation by these low-cost, high-throughput technologies in genomics is being matched by that of other technologies, such as real-time imaging and mass spectrometry-based flow cytometry. Success in the life sciences will depend on our ability to properly interpret the large-scale, high-dimensional data sets that are generated by these technologies, which in turn requires us to adopt advances in informatics. Here we discuss how we can master the different types of computational environments that exist – such as cloud and heterogeneous computing – to successfully tackle our big data problems.


July 7, 2019

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.


July 7, 2019

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.


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