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July 7, 2019  |  

Prognostic significance of novel katG mutations in Mycobacterium tuberculosis

By using whole genome sequencing (WGS), researchers are beginning to understand the genetic diversity of Mycobacterium tuberculosis (MTB) and its consequences for the diagnosis of multidrug-resistant tuberculosis (MDR–TB) on a genomic scale. The Global Consortium for Drug-resistant TB Diagnostics (GCDD) conducted a genome scale variant analyses of 366 clinical MTB genomes (mostly MDR/XDR [extensively drug resistant]) from four countries in order to inform the development of rapid molecular diagnostics. This project has been extended by performing an evolutionary analysis of isoniazid (INH)-resistant isolates for prognostic purposes.


July 7, 2019  |  

CHOgenome.org 2.0: Genome resources and website updates.

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


July 7, 2019  |  

A gapless, unambiguous genome sequence of the Enterohemorrhagic Escherichia coli O157:H7 strain EDL933.

Escherichia coli EDL933 is the prototypic strain for enterohemorrhagic E. coli serotype O157:H7, associated with deadly food-borne outbreaks. Because the publicly available sequence of the EDL933 genome has gaps and >6,000 ambiguous base calls, we here present an updated high-quality, unambiguous genome sequence with no assembly gaps. Copyright © 2014 Latif et al.


July 7, 2019  |  

Signature gene expression reveals novel clues to the molecular mechanisms of dimorphic transition in Penicillium marneffei.

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions.


July 7, 2019  |  

Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea.

TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.


July 7, 2019  |  

Cerulean: A hybrid assembly using high throughput short and long reads

Genome assembly using high throughput data with short reads, arguably, remains an unresolvable task in repetitive genomes, since when the length of a repeat exceeds the read length, it becomes difficult to unambiguously connect the flanking regions. The emergence of third generation sequencing (Pacific Biosciences) with long reads enables the opportunity to resolve complicated repeats that could not be resolved by the short read data. However, these long reads have high error rate and it is an uphill task to assemble the genome without using additional high quality short reads. Recently, Koren et al. 2012 proposed an approach to use high quality short reads data to correct these long reads and, thus, make the assembly from long reads possible. However, due to the large size of both dataset (short and long reads), error-correction of these long reads requires excessively high computational resources, even on small bacterial genomes. In this work, instead of error correction of long reads, we first assemble the short reads and later map these long reads on the assembly graph to resolve repeats.


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  |  

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  |  

Complete mitogenome of Indian mottled eel, Anguilla bengalensis bengalensis (Gray, 1831) through PacBio RSII sequencing.

Complete mitogenome sequence for Anguilla bengalensis bengalensis (family Anguillidae) was generated through third-generation sequencing platform. The 16?714 bp mitgenome sequence contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a non-coding (control) region. The gene order was identical to that observed in most of the other vertebrates. The comparison of complete mitogenome sequence of Indian mottled eel generated during this study with two other subspecies did not agree with the taxonomic status of the three subspecies and considered as one species.


July 7, 2019  |  

A genomic view of short tandem repeats.

Short tandem repeats (STRs) are some of the fastest mutating loci in the genome. Tools for accurately profiling STRs from high-throughput sequencing data have enabled genome-wide interrogation of more than a million STRs across hundreds of individuals. These catalogs have revealed that STRs are highly multiallelic and may contribute more de novo mutations than any other variant class. Recent studies have leveraged these catalogs to show that STRs play a widespread role in regulating gene expression and other molecular phenotypes. These analyses suggest that STRs are an underappreciated but rich reservoir of variation that likely make significant contributions to Mendelian diseases, complex traits, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.


July 7, 2019  |  

Neurotrophin biology at NGF 2016: From fundamental science to clinical applications.

In 1986, members of the growing neurotrophin community came together to honor the scientific contributions (and 77th birth- day) of Dr. Rita Levi-Montalcini. The celebration took the form of a conference dedicated to the field birthed by Dr. Levi-Montalcini’s discovery of nerve growth factor (NGF), for which she shared the Nobel Prize later that year with Stanley Cohen. The meeting proved to be a great success, and eventually became an ongoing series. The NGF 2016 meeting, held at the beautiful Asilomar conference cen- ter in Monterey, California, was the 13th meeting in this series, and marked the 30th anniversary of the original meeting. A diverse col- lection of investigators, representing academia and industry across 4 continents, gathered to celebrate the past 30 years, discuss the current state of the art, and share in the excitement of envisioning the next 30 years of neurotrophic factor research and applications.


July 7, 2019  |  

An amoebal grazer of cyanobacteria requires cobalamin produced by heterotrophic bacteria.

Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial foodwebs. Many amoebal species can be cultivated axenically in rich media or monoxenically with single bacterial prey species. Here we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using a defined non-redundant transposon library of Vibrio cholerae which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutants in V. cholerae and the Pseudomonas species coisolate do not support growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support growth of amoeba LPG3. Instead, we show that it requires cobalamin which is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial foodwebs.Importance. Cyanobacteria are important primary producers in aquatic environments where they are grazed upon by a variety of phagotrophic protists, and hence have an impact on nutrient flux at the base of microbial foodwebs. Here we characterize amoebal isolate LPG3 which consumes cyanobacteria as its primary food source but that also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by the heterotrophic bacteria, and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors which may shape microbial foodwebs while also providing insight into corrinoid specificity in eukaryotes. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

HapCUT2: robust and accurate haplotype assembly for diverse sequencing technologies.

Many tools have been developed for haplotype assembly-the reconstruction of individual haplotypes using reads mapped to a reference genome sequence. Due to increasing interest in obtaining haplotype-resolved human genomes, a range of new sequencing protocols and technologies have been developed to enable the reconstruction of whole-genome haplotypes. However, existing computational methods designed to handle specific technologies do not scale well on data from different protocols. We describe a new algorithm, HapCUT2, that extends our previous method (HapCUT) to handle multiple sequencing technologies. Using simulations and whole-genome sequencing (WGS) data from multiple different data types-dilution pool sequencing, linked-read sequencing, single molecule real-time (SMRT) sequencing, and proximity ligation (Hi-C) sequencing-we show that HapCUT2 rapidly assembles haplotypes with best-in-class accuracy for all data types. In particular, HapCUT2 scales well for high sequencing coverage and rapidly assembled haplotypes for two long-read WGS data sets on which other methods struggled. Further, HapCUT2 directly models Hi-C specific error modalities, resulting in significant improvements in error rates compared to HapCUT, the only other method that could assemble haplotypes from Hi-C data. Using HapCUT2, haplotype assembly from a 90× coverage whole-genome Hi-C data set yielded high-resolution haplotypes (78.6% of variants phased in a single block) with high pairwise phasing accuracy (~98% across chromosomes). Our results demonstrate that HapCUT2 is a robust tool for haplotype assembly applicable to data from diverse sequencing technologies.© 2017 Edge et al.; Published by Cold Spring Harbor Laboratory Press.


July 7, 2019  |  

The dynamic three-dimensional organization of the diploid yeast genome.

The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.


July 7, 2019  |  

Evolutionary strata on young mating-type chromosomes despite the lack of sexual antagonism.

Sex chromosomes can display successive steps of recombination suppression known as “evolutionary strata,” which are thought to result from the successive linkage of sexually antagonistic genes to sex-determining genes. However, there is little evidence to support this explanation. Here we investigate whether evolutionary strata can evolve without sexual antagonism using fungi that display suppressed recombination extending beyond loci determining mating compatibility despite lack of male/female roles associated with their mating types. By comparing full-length chromosome assemblies from five anther-smut fungi with or without recombination suppression in their mating-type chromosomes, we inferred the ancestral gene order and derived chromosomal arrangements in this group. This approach shed light on the chromosomal fusion underlying the linkage of mating-type loci in fungi and provided evidence for multiple clearly resolved evolutionary strata over a range of ages (0.9-2.1 million years) in mating-type chromosomes. Several evolutionary strata did not include genes involved in mating-type determination. The existence of strata devoid of mating-type genes, despite the lack of sexual antagonism, calls for a unified theory of sex-related chromosome evolution, incorporating, for example, the influence of partially linked deleterious mutations and the maintenance of neutral rearrangement polymorphism due to balancing selection on sexes and mating types.


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