ResearchPad - genomic-methodologies:-genome-variation-detection https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples]]> https://www.researchpad.co/article/N9c129733-73ca-419a-a4e4-cc8ba9c6cd17 Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.

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<![CDATA[Identification of <i>Acinetobacter baumannii</i> loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with <i>Kaptive</i> ]]> https://www.researchpad.co/article/Nf6eaf949-ff81-4776-8532-0dc36d9271e2 Multiply antibiotic-resistant infections are a global public health concern and accurate tracking of the spread of specific lineages is needed. Variation in the composition and structure of capsular polysaccharide (CPS), a critical determinant of virulence and phage susceptibility, makes it an attractive epidemiological marker. The outer core (OC) of lipooligosaccharide also exhibits variation. To take better advantage of the untapped information available in whole genome sequences, we have created a curated reference database of 92 publicly available gene clusters at the locus encoding proteins responsible for biosynthesis and export of CPS (K locus), and a second database for 12 gene clusters at the locus for outer core biosynthesis (OC locus). Each entry has been assigned a unique KL or OCL number, and is fully annotated using a simple, transparent and standardized nomenclature. These databases are compatible with Kaptive, a tool for in silico typing of bacterial surface polysaccharide loci, and their utility was validated using (a) >630 assembled draft genomes for which the KL and OCL regions had been previously typed manually, and (b) 3386 genome assemblies downloaded from NCBI. Among the previously typed genomes, Kaptive was able to confidently assign KL and OCL types with 100 % accuracy. Among the genomes retrieved from NCBI, Kaptive detected known KL and OCL in 87 and 90 % of genomes, respectively, indicating that the majority of common KL and OCL types are captured within the databases; 13 of the 92 KL in the database were not detected in any publicly available whole genome assembly. The failure to assign a KL or OCL type may indicate incomplete or poor-quality genomes. However, further novel variants may remain to be documented. Combining outputs with multilocus sequence typing (Institut Pasteur scheme) revealed multiple KL and OCL types in collections of a single sequence type (ST) representing each of the two predominant globally distributed clones, ST1 of GC1 and ST2 of GC2, and in collections of other clones comprising >20 isolates each (ST10, ST25, and ST140), indicating extensive within-clone replacement of these loci. The databases are available at https://github.com/katholt/Kaptive and will be updated as further locus types become available.

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<![CDATA[DEN-IM: dengue virus genotyping from amplicon and shotgun metagenomic sequencing]]> https://www.researchpad.co/article/Naff780e0-abf1-4148-a63f-d2882aa976e3 Dengue virus (DENV) represents a public health threat and economic burden in affected countries. The availability of genomic data is key to understanding viral evolution and dynamics, supporting improved control strategies. Currently, the use of high-throughput sequencing (HTS) technologies, which can be applied both directly to patient samples (shotgun metagenomics) and to PCR-amplified viral sequences (amplicon sequencing), is potentially the most informative approach to monitor viral dissemination and genetic diversity by providing, in a single methodological step, identification and characterization of the whole viral genome at the nucleotide level. Despite many advantages, these technologies require bioinformatics expertise and appropriate infrastructure for the analysis and interpretation of the resulting data. In addition, the many software solutions available can hamper the reproducibility and comparison of results. Here we present DEN-IM, a one-stop, user-friendly, containerized and reproducible workflow for the analysis of DENV short-read sequencing data from both amplicon and shotgun metagenomics approaches. It is able to infer the DENV coding sequence (CDS), identify the serotype and genotype, and generate a phylogenetic tree. It can easily be run on any UNIX-like system, from local machines to high-performance computing clusters, performing a comprehensive analysis without the requirement for extensive bioinformatics expertise. Using DEN-IM, we successfully analysed two types of DENV datasets. The first comprised 25 shotgun metagenomic sequencing samples from patients with variable serotypes and genotypes, including an in vitro spiked sample containing the four known serotypes. The second consisted of 106 paired-end and 76 single-end amplicon sequences of DENV 3 genotype III and DENV 1 genotype I, respectively, where DEN-IM allowed detection of the intra-genotype diversity. The DEN-IM workflow, parameters and execution configuration files, and documentation are freely available at https://github.com/B-UMMI/DEN-IM).

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<![CDATA[Optimization of sample preparation for culture-independent sequencing of <i>Bordetella pertussis</i> ]]> https://www.researchpad.co/article/Ncc9bf199-c3fe-43dc-a79c-038b91a7ae78 , the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600 000 deaths are prevented annually by pertussis vaccination, but infection was still responsible for over 63 000 deaths globally in 2013. The re-emergence of has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling . Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target . However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target DNA. Sequencing read depth improved five-fold in reads to . Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of in the era of culture-independent testing.

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