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April 21, 2020

Molecular Epidemiology of Candida auris in Colombia Reveals a Highly Related, Countrywide Colonization With Regional Patterns in Amphotericin B Resistance.

Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia.We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates.Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance.Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

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April 21, 2020

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected.To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms.The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing.A novel IncZ-7 plasmid pEC013 (~118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013.The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid’s heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety “Finola.” The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ~40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant. © 2019 Laverty et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020

Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ~24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel’s original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

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April 21, 2020

Assessment of the microbial diversity of Chinese Tianshan tibicos by single molecule, real-time sequencing technology.

Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains.

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April 21, 2020

Construction of full-length Japanese reference panel of class I HLA genes with single-molecule, real-time sequencing.

Human leukocyte antigen (HLA) is a gene complex known for its exceptional diversity across populations, importance in organ and blood stem cell transplantation, and associations of specific alleles with various diseases. We constructed a Japanese reference panel of class I HLA genes (ToMMo HLA panel), comprising a distinct set of HLA-A, HLA-B, HLA-C, and HLA-H alleles, by single-molecule, real-time (SMRT) sequencing of 208 individuals included in the 1070 whole-genome Japanese reference panel (1KJPN). For high-quality allele reconstruction, we developed a novel pipeline, Primer-Separation Assembly and Refinement Pipeline (PSARP), in which the SMRT sequencing and additional short-read data were used. The panel consisted of 139 alleles, which were all extended from known IPD-IMGT/HLA sequences, contained 40 with novel variants, and captured more than 96.5% of allelic diversity in 1KJPN. These newly available sequences would be important resources for research and clinical applications including high-resolution HLA typing, genetic association studies, and analyzes of cis-regulatory elements.

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April 21, 2020

Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai, China, 2006-2016.

Colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella. Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1-harbouring Salmonella in community-acquired infections should be estimated.This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006-2016. Molecular characteristics of the mcr-1-positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes.Among the 12,053 Salmonella isolates, 37 mcr-1-harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1-harbouring strains were aged <5?years. All strains, including fluoroquinolone-resistant and/or extended-spectrum ß-lactamase-producing strains, were multi-drug resistant. S. Typhimurium had higher mcr-1 plasmid acquisition ability compared with other common serovars. Phylogeny based on the genomes combined with complete plasmid sequences revealed some clusters, suggesting the presence of mcr-1-harbouring Salmonella outbreaks in the community. Most mcr-1-positive strains were clustered together with the pork strains, strongly suggesting pork consumption as a main infection source.The mcr-1-harbouring Salmonella prevalence in community-acquired diarrhoea displays a rapid increase trend, and the ESBL-mcr-1-harbouring Salmonella poses a threat for children. These findings highlight the necessary and significance of prohibiting colistin use in animals and continuous monitoring of mcr-1-harbouring Salmonella.Copyright © 2019. Published by Elsevier B.V.

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April 21, 2020

The role of genomic structural variation in the genetic improvement of polyploid crops

Many of our major crop species are polyploids, containing more than one genome or set of chromosomes. Polyploid crops present unique challenges, including difficulties in genome assembly, in discriminating between multiple gene and sequence copies, and in genetic mapping, hindering use of genomic data for genetics and breeding. Polyploid genomes may also be more prone to containing structural variation, such as loss of gene copies or sequences (presence–absence variation) and the presence of genes or sequences in multiple copies (copy-number variation). Although the two main types of genomic structural variation commonly identified are presence–absence variation and copy-number variation, we propose that homeologous exchanges constitute a third major form of genomic structural variation in polyploids. Homeologous exchanges involve the replacement of one genomic segment by a similar copy from another genome or ancestrally duplicated region, and are known to be extremely common in polyploids. Detecting all kinds of genomic structural variation is challenging, but recent advances such as optical mapping and long-read sequencing offer potential strategies to help identify structural variants even in complex polyploid genomes. All three major types of genomic structural variation (presence–absence, copy-number, and homeologous exchange) are now known to influence phenotypes in crop plants, with examples of flowering time, frost tolerance, and adaptive and agronomic traits. In this review, we summarize the challenges of genome analysis in polyploid crops, describe the various types of genomic structural variation and the genomics technologies and data that can be used to detect them, and collate information produced to date related to the impact of genomic structural variation on crop phenotypes. We highlight the importance of genomic structural variation for the future genetic improvement of polyploid crops.

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April 21, 2020

Genome of the Komodo dragon reveals adaptations in the cardiovascular and chemosensory systems of monitor lizards.

Monitor lizards are unique among ectothermic reptiles in that they have high aerobic capacity and distinctive cardiovascular physiology resembling that of endothermic mammals. Here, we sequence the genome of the Komodo dragon Varanus komodoensis, the largest extant monitor lizard, and generate a high-resolution de novo chromosome-assigned genome assembly for V. komodoensis using a hybrid approach of long-range sequencing and single-molecule optical mapping. Comparing the genome of V. komodoensis with those of related species, we find evidence of positive selection in pathways related to energy metabolism, cardiovascular homoeostasis, and haemostasis. We also show species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and other lizard lineages. Together, these evolutionary signatures of adaptation reveal the genetic underpinnings of the unique Komodo dragon sensory and cardiovascular systems, and suggest that selective pressure altered haemostasis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. The Komodo dragon genome is an important resource for understanding the biology of monitor lizards and reptiles worldwide.

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April 21, 2020

PacBio sequencing reveals bacterial community diversity in cheeses collected from different regions.

Cheese is a fermented dairy product that is popular for its unique flavor and nutritional value. Recent studies have shown that microorganisms in cheese play an important role in the fermentation process and determine the quality of the cheese. We collected 12 cheese samples from different regions and studied the composition of their bacterial communities using PacBio small-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA). Our data revealed 144 bacterial genera (including Lactobacillus, Streptococcus, Lactococcus, and Staphylococcus) and 217 bacterial species (including Lactococcus lactis, Streptococcus thermophilus, Staphylococcus equorum, and Streptococcus uberis). We investigated the flavor quality of the cheese samples using an electronic nose system and we found differences in flavor-quality indices among samples from different regions. We found a clustering tendency based on flavor quality using principal component analysis. We found correlations between lactic acid bacteria and the flavor quality of the cheese samples. Biodegradation and metabolism of xenobiotics, and lipid-metabolism-related pathways, were predicted to contribute to differences in cheese flavor using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). This preliminary study explored the bacterial communities in cheeses collected from different regions and their potential genome functions from the perspective of flavor quality.Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

A survey and evaluations of histogram-based statistics in alignment-free sequence comparison.

Since the dawn of the bioinformatics field, sequence alignment scores have been the main method for comparing sequences. However, alignment algorithms are quadratic, requiring long execution time. As alternatives, scientists have developed tens of alignment-free statistics for measuring the similarity between two sequences.We surveyed tens of alignment-free k-mer statistics. Additionally, we evaluated 33 statistics and multiplicative combinations between the statistics and/or their squares. These statistics are calculated on two k-mer histograms representing two sequences. Our evaluations using global alignment scores revealed that the majority of the statistics are sensitive and capable of finding similar sequences to a query sequence. Therefore, any of these statistics can filter out dissimilar sequences quickly. Further, we observed that multiplicative combinations of the statistics are highly correlated with the identity score. Furthermore, combinations involving sequence length difference or Earth Mover’s distance, which takes the length difference into account, are always among the highest correlated paired statistics with identity scores. Similarly, paired statistics including length difference or Earth Mover’s distance are among the best performers in finding the K-closest sequences. Interestingly, similar performance can be obtained using histograms of shorter words, resulting in reducing the memory requirement and increasing the speed remarkably. Moreover, we found that simple single statistics are sufficient for processing next-generation sequencing reads and for applications relying on local alignment. Finally, we measured the time requirement of each statistic. The survey and the evaluations will help scientists with identifying efficient alternatives to the costly alignment algorithm, saving thousands of computational hours.The source code of the benchmarking tool is available as Supplementary Materials. © The Author 2017. Published by Oxford University Press.

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April 21, 2020

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

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April 21, 2020

Effect of sulfur-iron modified biochar on the available cadmium and bacterial community structure in contaminated soils.

Cadmium contamination in paddy soils has aroused increasing concern around the world, and biochar has many positive properties, such as large specific surface areas, micro porous structure for the heavy metal immobilization in soils. However there are few studies on sulfur-iron modified biochar as well as its microbiology effects. The purpose of this study was to evaluate the Cd immobilization effects of sulfur or sulfur-iron modified biochar and its related microbial community changes in Cd-contaminated soils. SEM-EDX analysis confirmed that sulfur and iron were loaded on the raw biochar successfully. Sulfur-modified biochar (S-BC) and sulfur-iron modified biochar (SF-BC) addition increased pH value and the content of soil organic matter, and also decreased DTPA-extractable Cd. There was a negative significant correlation between organic matter content and the available Cd (P?

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April 21, 2020

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5?kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15?megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

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