Abstract
ABSTRACTThis first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.
Highlights
106 Whole genome sequencing (WGS) of SARS-CoV-2 isolates has been used in many countries mainly to determine (i) specific viral lineages and (ii) the molecular epidemiological context
external quality assessments (EQAs) can 122 ensure and improve testing quality and results comparability. They allow, if sufficiently scaled, the comparison of test performance of in-house developed and commercial assays. 125 To date, no EQA results have been published focusing on WGS of SARS-CoV-2, 126 some publications have shared quality aspects of single centre’s experiences [2,3]
Centres 7 and 9 are on 205 the lower end of the spectrum (mean depth of 325x 275 (SD) and 313x 132, respectively), whereas all other labs usually sequenced to a mean depth between 1000x and 8000x. 208 The majority of samples could be assembled to a consensus genome by all centres with the exception of NGS8 for which assembly failed partially for centre 7 and completely failed for centre 9 as seen by the percentage of missing data shown in figure 1B. 213 SNPs and Indels Variants have been assessed as reported and are displayed in Supplementary Figure S1A-J as a dot plot indicating presence and absence of the variant
Summary
106 Whole genome sequencing (WGS) of SARS-CoV-2 isolates has been used in many countries mainly to determine (i) specific viral lineages and (ii) the molecular epidemiological context. External quality assessments (EQAs) represent a corner stone in introducing new test methods, capacity building, and ensuring a baseline quality level This is even more important in a pandemic situation, when a novel, previously unknown pathogen necessitates prompt development, validation and roll out of assays for which microbiological expertise and diagnostic knowledge are limited. In this context, EQAs can ensure and improve testing quality and results comparability. 125 To date, no EQA results have been published focusing on WGS of SARS-CoV-2, some publications have shared quality aspects of single centre’s experiences [2,3] Along, these lines, individual centres in Switzerland have published protocols on WGS with 128 different epidemiological questions [4,5].
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