A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Kathy E Raven,Sharon J Peacock,Beth Blane,Nicholas Brown,Danielle Leek,Sophia T Girgis,Asha Akram

doi:10.1038/s41598-020-80031-8

Abstract

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Highlights

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories
Ten of these panel 4 isolates were associated with three putative outbreaks (E. faecium (n = 3), E. cloacae (n = 3) and S. aureus (n = 4)), and six panel 4 isolates contained specific genes encoding antibiotic resistance, or capsular type (Neisseria meningitidis (n = 3))
We sought to develop a single method for extracting DNA from multiple different bacterial species direct from colonies on solid media, prior to a common protocol for library preparation and whole genome sequencing using the Illumina Nextera Flex kit

Summary

Introduction

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories. Local and regional clinical microbiology laboratories that adopt sequencing technologies to support hospital outbreak investigation and prescribing decisions will face relatively low sample numbers and the requirement to sequence multiple different species in the same sequencing run to minimise turnaround time and costs. Once DNA has been extracted, the same methodology can be used across different bacterial species to prepare and sequence DNA libraries and the only adjustment required is to determine how many isolates of any given species can be included in a single run based on genome size. Analysis of the sequence data generated corroborated the findings of previous results for 16 clinical isolates belonging to 7 different pathogenic species associated with transmission, outbreaks and multidrug resistance

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