An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

Abstract

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. By optimising a logistics pipeline that collected isolates as thermolysates, permitting shipment in ambient conditions, the project assembled a diverse collection of 10,419 clinical and environmental isolates from low- and middle-income countries in less than one year. The bacteria were obtained from 51 countries/territories dating from 1949 to 2017, with a focus on Africa and Latin-America. All isolates were collected in barcoded tubes and genome sequenced using an optimised DNA extraction method and the LITE pipeline for library construction. After Illumina sequencing, the total reagent cost was less than USD$10 per genome. Our method can be applied to genome-sequence other large bacterial collections at a relatively low cost, within a limited timeframe, to support global collaborations.

Genomic diversity and antimicrobial resistance among non-typhoidal Salmonella associated with human disease in The Gambia

“…Libraries were prepared using the Nextera XT kit using the Illumina MiSeq system. The DNA extraction and sequencing of samples processed at the University of Liverpool were carried out using an optimised method for large-scale sequencing [21], including the bespoke LITE (Low Input, Transposase Enabled) pipeline for library construction, and Illumina HiSeq sequencing technology. Both sites used the 2×150 bp read protocol.…”

Complete Genome Sequences of African Salmonella enterica Serovar Enteritidis Clinical Isolates Associated with Bloodstream Infection

“…Long-read sequencing was performed by the Centre for Genomic Research (University of Liverpool, UK) in a PacBio single-molecule real-time (SMRT) cell (P6/C4 chemistry) using SMRTbell Template v1.0 (Pacific Biosciences; catalog number 100-259-100) library preparation with g-TUBE (Covaris) fragmentation and size selection of 15 to 50 kb with 0.75% agarose cassette (BluePippin; catalog number BMF7510). Illumina HiSeq sequencing was performed as part of the 10KSG project ( 11 ) and by MicrobesNG (UK) using the Nextera XT library prep kit (Illumina, USA) with modifications (2 ng DNA and 1 min PCR elongation) and 250-bp paired-end protocol. The reads were adapter trimmed using Trimmomatic v0.30, with a sliding window quality cutoff of Q15 ( 12 ).…”

A Genomic Census of the Chicken Gut Microbiome using Metagenomics and Culture

“…We initially analysed the faecal samples using the k-mer-based program Kraken 2, followed by re ned phylogenetic analysis using the allied program Bracken [17] (Table S3). Unsurprisingly, these programs assigned sequence reads from the faecal samples to all three domains of life, as well as to viruses (Fig.…”

“…The remaining dataset underwent taxonomic pro ling using Kraken 2 against a microbial database built from all complete/representative archaeal, bacterial, fungal, protozoan, viral and UniVec_Core sequences in RefSeq [53] in January 2020. Bracken [17] was used to estimate taxon abundance from the Kraken 2 pro les, accepting only those taxa with ≥1000 assigned reads. Bracken-database les were generated using "bracken-build" on our microbial database and visualised using KronaTools [54].…”

“…To estimate species richness and diversity, the Phyloseq package of R [74] was applied to the output from Bracken [17] on all of our chicken faecal metagenomic datasets. The six faecal samples that showed highest species richness and taxonomic diversity were selected for culture-based studies.…”