Schematic overview of the design, workflow, and technologies adopted in this study with their hands-on and sequencing turnaround time. The performance was studied by comparing genome coverage, read depth, amplicon distribution, variant calling, and the proportion of on-target reads. 1) employing a diversity of sequencers with a wide range of throughput, accuracy, and runtime were compared using clinical samples. Here, we describe a cross-platform benchmark study that includes Illumina, Ion Torrent, and nanopore-based SARS-CoV-2 sequencing technologies in one study. A recent external quality assessment (EQA) report assessed the outcome of complete workflows from nucleic acid extraction to the reported consensus sequence by testing SARS-CoV-2 cultured isolates however, no detailed distinction between the different workflow components could be made. In contrast, cross-platform studies including short and long read sequencing platforms and metagenomics remain relatively scarce and limited to a maximum of two different sequence platforms. Benchmark studies of SARS-CoV-2 genome sequencing technologies are limited and generally restricted to comparison of protocols for the single type of sequencing technology available at the study site of the authors – or cross-platform studies limited to only amplicon sequencing protocols –. However, these studies have been typically focused on the technology developed by the authors, whereas comparison of a novel protocol with other methods is limited. Studies have been published on SARS-CoV-2 WGS with innovative protocol adaptations in order to decrease the error rate and the turnaround time by combining PCR and tagging steps. SARS-CoV-2 whole genome sequencing (WGS) protocols have been improved to increase the technical performance, including sensitivity and genome coverage, and logistical aspects have also been addressed, such as scalability and hands-on time –. Ī wide range of SARS-CoV-2 next-generation sequencing (NGS) technologies and protocols have been developed and adapted since the first genome sequence was generated using a metagenomic approach –. The widespread application of genomics for pandemic surveillance is exemplified by more than 15 million SARS-CoV-2 sequences deposited in the GISAID repository as of February 2023.
In addition, genomic surveillance assists in contact tracing, transmission tracking at population level, and public-health decision-making. Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven critical for early detection of the rise and spread of SARS-CoV-2 variants of concern, for monitoring and developing effective diagnostic, therapeutic, and preventive strategies –. This study provides data that assist laboratories when selecting protocols for their specific setting. In conclusion, the studied protocols differed on a variety of the studied metrics. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log 10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Correlation of coverage with PCR Ct values varied per protocol. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies.
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