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July 7, 2019

Serinibacter

The genus Serinibacter belongs, based on the phylogenetic analysis of the nearly full-length 16S rRNA gene, to the Beutenbergiaceae together with the genera Beutenbergia, Salana, and Miniimonas. The two species of the genus Serinibacter shared 99.6% 16S rRNA gene sequence similarity but low DNA DNA relatedness. Cells are irregular rods, Gram-stain positive, not acid-fast. Endospores are not formed. Nonmotile. Aerobic to anaerobic. Oxidase-negative, catalase-positive. The peptidoglycan type is A4a with an l-Ser residue at position 1 of the peptide subunit. The acyl type is acetyl. The major cell-wall sugar is galactose. The predominant menaquinone is MK-8(H4). The major polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and unidentified phospholipids. Phosphatidylethanolamine is absent. The cellular fatty acid profile is dominated by the occurrence of iso- and anteiso-branched-chain acids. Mycolic acids are absent. The genomic G+C content is 70.7 to 72.8 mol%.

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July 7, 2019

Genomic insights into a sustained national outbreak of Yersinia pseudotuberculosis.

In 2014, a sustained outbreak of yersiniosis due to Yersinia pseudotuberculosis occurred across all major cities in New Zealand (NZ), with a total of 220 laboratory-confirmed cases, representing one of the largest ever reported outbreaks of Y. pseudotuberculosis. Here, we performed whole genome sequencing of outbreak-associated isolates to produce the largest population analysis to date of Y. pseudotuberculosis, giving us unprecedented capacity to understand the emergence and evolution of the outbreak clone. Multivariate analysis incorporating our genomic and clinical epidemiological data strongly suggested a single point-source contamination of the food chain, with subsequent nationwide distribution of contaminated produce. We additionally uncovered significant diversity in key determinants of virulence, which we speculate may help explain the high morbidity linked to this outbreak.

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July 7, 2019

Microbial metagenomics mock scenario-based sample simulation (M3S3).

Shotgun sequencing in increasingly applied in clinical microbiology for unbiased culture-independent diagnosis. While software solutions for metagenomics proliferate, integration of metagenomics in clinical care, requires method standardisation and validation. Virtual metagenomics samples could underpin validation by substituting real samples and thus we sought to develop a novel solution for simulation of metagenomics samples based on user-defined clinical scenarios.We designed the Microbial Metagenomics Mock Scenario-based Sample Simulation (M3S3) workflow, which allows users to generate virtual samples from raw reads or assemblies. The M3S3 output is a mock sample in FASTQ or FASTA format. M3S3 was tested by generating virtual samples for ten challenging infectious disease scenarios, involving a background matrix ‘spiked’ in silico with pathogens including mixtures. Replicate samples (seven per scenario) were used to represent different compositional ratios. Virtual samples were analysed using Taxonomer and Kraken db.The ten challenge scenarios were successfully applied, generating 80 samples. For all tested scenarios, the virtual samples showed sequence compositions as predicted from the user input. Spiked pathogen sequences were identified with the majority of the replicates and most exhibited acceptable abundance (deviation between expected and observed abundance of spiked pathogens), with slight differences observed between software tools.Despite demonstrated proof-of-concept, integration of clinical metagenomics in routine microbiology remains a substantial challenge. M3S3 is capable of producing virtual samples on-demand, simulating a spectrum of clinical diagnostic scenarios of varying complexity. The M3S3 tool can therefore support the development and validation of standardised metagenomics applications. Copyright © 2017. Published by Elsevier Ltd.

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July 7, 2019

A high throughput screen for active human transposable elements.

Transposable elements (TEs) are mobile genetic sequences that randomly propagate within their host’s genome. This mobility has the potential to affect gene transcription and cause disease. However, TEs are technically challenging to identify, which complicates efforts to assess the impact of TE insertions on disease. Here we present a targeted sequencing protocol and computational pipeline to identify polymorphic and novel TE insertions using next-generation sequencing: TE-NGS. The method simultaneously targets the three subfamilies that are responsible for the majority of recent TE activity (L1HS, AluYa5/8, and AluYb8/9) thereby obviating the need for multiple experiments and reducing the amount of input material required.Here we describe the laboratory protocol and detection algorithm, and a benchmark experiment for the reference genome NA12878. We demonstrate a substantial enrichment for on-target fragments, and high sensitivity and precision to both reference and NA12878-specific insertions. We report 17 previously unreported loci for this individual which are supported by orthogonal long-read evidence, and we identify 1470 polymorphic and novel TEs in 12 additional samples that were previously undocumented in databases of insertion polymorphisms.We anticipate that future applications of TE-NGS alongside exome sequencing of patients with sporadic disease will reduce the number of unresolved cases, and improve estimates of the contribution of TEs to human genetic disease.

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July 7, 2019

FDA-CDC antimicrobial resistance isolate bank: A publicly-available resource to support research, development and regulatory requirements.

The FDA-CDC Antimicrobial Resistance Isolate Bank was created in July 2015 as a publicly available resource to combat antimicrobial resistance. It is a curated repository of bacterial isolates with an assortment of clinically-important resistance mechanisms that have been phenotypically and genotypically characterized. In the first two years of operation, the Bank offered 14 panels comprising 496 unique isolates and had filled 486 orders from 394 institutions throughout the United States. New panels are being added. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

De novo mutations resolve disease transmission pathways in clonal malaria

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However, these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here, we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a combination of sequencing, library preparation, and genotyping methods that have been optimized for accuracy in low-complexity genomic regions, we have detected de novo mutations that distinguish nominally identical parasites from clonal lineages. Despite its slower evolutionary rate compared with bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2?years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

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July 7, 2019

Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.

Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.

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July 7, 2019

Construction of two whole genome radiation hybrid panels for dromedary (Camelus dromedarius): 5000RAD and 15000RAD.

The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000RADand 15000RAD) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000RADpanel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000RADpanel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000RADpanel is an important high-resolution complement to the main 5000RADpanel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.

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July 7, 2019

Complete genome sequence of Escherichia coli 81009, a representative of the sequence type 131 C1-M27 clade with a multidrug-resistant phenotype.

The sequence type 131 (ST131)-H30 clone is responsible for a significant proportion of multidrug-resistant extraintestinal Escherichia coli infections. Recently, the C1-M27 clade of ST131-H30, associated with blaCTX-M-27, has emerged. The complete genome sequence of E. coli isolate 81009 belonging to this clone, previously used during the development of ST131-specific monoclonal antibodies, is reported here. Copyright © 2018 Mutti et al.

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July 7, 2019

Complete genome sequence of a type strain of Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus complex.

Mycobacterium abscessus subsp. bolletii is a rapidly growing mycobacterial organism for which the taxonomy is unclear. Here, we report the complete genome sequence of a Mycobacterium abscessus subsp. bolletii type strain. This sequence will provide essential information for future taxonomic and comparative genome studies of these mycobacteria.

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July 7, 2019

A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

During DNA extraction the DNA molecule undergoes physical and chemical shearing, causing the DNA to fragment into shorter and shorter pieces. Under common laboratory conditions this fragmentation yields DNA fragments of 5-35 kilobases (kb) in length. This fragment length is more than sufficient for DNA sequencing using short-read technologies which generate reads 50-600 bp in length, but insufficient for long-read sequencing and linked reads where fragment lengths of more than 40 kb may be desirable. This study provides a theoretical framework for quality management to ensure access to high molecular weight DNA in samples. Shearing can be divided into physical and chemical shearing which generate different patterns of fragmentation. Exposure to physical shearing creates a characteristic fragment length where DNA fragments are cut in half by shear stress. This characteristic length can be measured using gel electrophoresis or instruments for DNA fragment analysis. Chemical shearing generates randomly distributed fragment lengths visible as a smear of DNA below the peak fragment length. By measuring the peak of DNA fragment length and the proportion of very short DNA fragments both sources of shearing can be measured using commonly used laboratory techniques, providing a suitable quantification of DNA integrity of DNA for sequencing with long-read technologies.

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July 7, 2019

Development of molecular markers linked to powdery mildew resistance GenePm4bby combining SNP discovery from transcriptome sequencing data with bulked segregant analysis (BSR-Seq) in wheat.

Powdery mildew resistance genePm4b, originating fromTriticum persicum, is effective against the prevalentBlumeria graminisf. sp.tritici(Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification ofPm4bduring the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-Seq technique and the bulked segregant analysis (BSR-Seq) was applied in an F2:3mapping population (237 families) derived from a pair of isogenic lines VPM1/7*Bainong 3217 F4(carryingPm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-Seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8 and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, 46 variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. FourPm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking thePm4bgene. Three SSR markers,Xics13,Xics43, andXics76, were incorporated in the new genetic linkage map, which locatedPm4bin a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a collinear relationship withBrachypodium distachyonchromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included C2 domain protein, peroxidase activity protein, protein kinases of PKc_like super family, Mlo family protein, and catalytic domain of the serine/threonine kinases (STKc_IRAK like super family). The markers developed in the present study facilitate identification ofPm4bduring its MAS practice.

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July 7, 2019

Supergene evolution triggered by the introgression of a chromosomal inversion.

Supergenes are groups of tightly linked loci whose variation is inherited as a single Mendelian locus and are a common genetic architecture for complex traits under balancing selection [1-8]. Supergene alleles are long-range haplotypes with numerous mutations underlying distinct adaptive strategies, often maintained in linkage disequilibrium through the suppression of recombination by chromosomal rearrangements [1, 5, 7-9]. However, the mechanism governing the formation of supergenes is not well understood and poses the paradox of establishing divergent functional haplotypes in the face of recombination. Here, we show that the formation of the supergene alleles encoding mimicry polymorphism in the butterfly Heliconius numata is associated with the introgression of a divergent, inverted chromosomal segment. Haplotype divergence and linkage disequilibrium indicate that supergene alleles, each allowing precise wing-pattern resemblance to distinct butterfly models, originate from over a million years of independent chromosomal evolution in separate lineages. These “superalleles” have evolved from a chromosomal inversion captured by introgression and maintained in balanced polymorphism, triggering supergene inheritance. This mode of evolution involving the introgression of a chromosomal rearrangement is likely to be a common feature of complex structural polymorphisms associated with the coexistence of distinct adaptive syndromes. This shows that the reticulation of genealogies may have a powerful influence on the evolution of genetic architectures in nature. Copyright © 2018 Elsevier Ltd. All rights reserved.

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