Evaluation of Rapid Library Preparation Protocols for Whole Genome Sequencing Based Outbreak Investigation

Helena M B Seth-Smith; Ferdinando Bonfiglio; Aline Cuénod; Josiane Reist; Adrian Egli; Daniel Wüthrich

doi:10.3389/fpubh.2019.00241

Evaluation of Rapid Library Preparation Protocols for Whole Genome Sequencing Based Outbreak Investigation

Front Public Health. 2019 Aug 27:7:241. doi: 10.3389/fpubh.2019.00241. eCollection 2019.

Authors

Helena M B Seth-Smith^{1

2

3}, Ferdinando Bonfiglio^{2

4}, Aline Cuénod^{1

2}, Josiane Reist², Adrian Egli^{1

2}, Daniel Wüthrich^{1

2

3}

Affiliations

¹ Division of Clinical Bacteriology and Mycology, University Hospital Basel, Basel, Switzerland.
² Applied Microbiology Research, Department of Biomedicine, University of Basel, Basel, Switzerland.
³ DBM Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Basel, Switzerland.
⁴ Personalized Health Basel, University of Basel, Basel, Switzerland.

Abstract

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 and time required 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: Illumina; NGS; bacteria; comparison; library; next generation sequencing; prokaryotes; whole genome sequencing.