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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Public Health | doi: 10.3389/fpubh.2019.00241

Evaluation of rapid library preparation protocols for whole genome sequencing based outbreak investigation

  • 1Division of Clinical Microbiology, University Hospital of Basel, Switzerland
  • 2Applied Microbiology Research, Department of Biomedicine, Faculty of Medicine, University of Basel, Switzerland
  • 3Swiss Institute of Bioinformatics (SIB), Switzerland
  • 4Personalized Health Basel, University of Basel, Switzerland

Whole genome sequencing (WGS) has become the new gold standard for bacterial outbreak investigation, due to the high resolution available for typing. While sequencing is currently predominantly performed on Illumina devices, the preceding library preparation can be performed using various protocols. Enzymatic fragmentation library preparation protocols are fast, have minimal hands-on time, and work with small quantities of DNA. The aim of our study was to compare three library preparation protocols for molecular typing: Nextera XT (Illumina); Nextera Flex (Illumina); and QIAseq FX (Qiagen). We selected 12 ATCC strains from human Gram-positive and Gram-negative pathogens with %G+C-content ranging from 27 % (Fusobacterium nucleatum) to 73 % (Micrococcus luteus), each having a high quality complete genome assembly available, to allow in-depth analysis of the resulting Illumina sequence data quality. Additionally, we selected isolates from previously analyzed cases of vancomycin-resistant Enterococcus faecium (VRE) (n=7) and a local outbreak of Klebsiella aerogenes (n=5). The number of protocol steps, time required and list prices were compared, in order to test the suitability for routine laboratory work. Data analyses were performed with standard tools commonly used in outbreak situations: Ridom SeqSphere+ for cgMLST; CLC genomics workbench for SNP analysis; and open source programs. Nextera Flex and QIAseq FX were found to be less sensitive than Nextera XT to variable %G+C-content, resulting in an almost uniform distribution of read-depth. Therefore, low coverage regions are reduced to a minimum resulting in a more complete representation of the genome. Thus, with these two protocols, more alleles were detected in the cgMLST analysis, producing a higher resolution of closely related isolates. Furthermore, they result in a more complete representation of accessory genes. In particular, the high data quality and relative simplicity of the workflow of Nextera Flex stood out in this comparison. This thorough comparison within an ISO/IEC 17025 accredited environment will be of interest to those aiming to optimize their clinical microbiological genome sequencing.

Keywords: NGS - next generation sequencing, Library, Illumina, Whole genome sequencing (WGS), comparison, Bacteria, prokaryotes

Received: 09 Apr 2019; Accepted: 12 Aug 2019.

Edited by:

Vitali Sintchenko, University of Sydney, Australia

Reviewed by:

Avram Levy, University of Western Australia, Australia
Rebecca Rockett, University of Sydney, Australia  

Copyright: © 2019 Seth-Smith, Bonfiglio, Cuénod, Reist, Egli and Wüthrich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Daniel Wüthrich, University Hospital of Basel, Division of Clinical Microbiology, Basel, 4056, Basel-Stadt, Switzerland,