Multiple approaches for massively parallel sequencing of SARS-CoV-2 genomes directly from clinical samples

Abstract

Background: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. Methods: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner.

Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection

“…We observed that the RT-LAMP assay was less sensitive and informative than multiplex PCR-based NGS in our study and other literature (20)(21)(22)(23)(24). NGS is a robust tool for obtaining extensive genetic information, allowing LOD values as low as 10 copies/ml for SARS-CoV-2 and serving as a reference test for COVID-19, especially for those challenging samples with a low viral content (2,(22)(23)(24). However, several experimental issues, such as erroneous barcode sequencing, the production of primer dimers, and potential cross-contamination between runs, may complicate sequence-based analyses and impact the validity of NGS results (25,26).…”

“…We observed that the RT-LAMP assay was less sensitive and informative than multiplex PCR-based NGS in our study and other literature (20)(21)(22)(23)(24). NGS is a robust tool for obtaining extensive genetic information, allowing LOD values as low as 10 copies/ml for SARS-CoV-2 and serving as a reference test for COVID-19, especially for those challenging samples with a low viral content (2,(22)(23)(24).…”

Initial insights into the genetic epidemiology of SARS-CoV-2 isolates from Kerala suggest local spread from limited introductions

“…The availability of high-throughput sequencing approaches has enabled researchers to sequence genomes as the pandemic progressed in their respective countries. A number of methods have been adopted for rapid high throughput sequencing of SARS-CoV-2 including shotgun sequencing [4] , PCR amplicon, and hybridization/capture-based enrichment and sequencing [5][6][7] .…”

“…A number of approaches have emerged for rapid and scalable sequencing of SARS-CoV-2 from clinical isolates. This includes direct shotgun approaches as well as targeted amplicon-based and targeted capture-based approaches [4][5][6] . Sequencing based approaches provide a unique opportunity for high fidelity of detection and for understanding the genetic epidemiology of SARS-CoV-2 [7] .…”

Within-patient genetic diversity of SARS-CoV-2

“…Viral isolation and sequencing efforts throughout the world have permitted the interrogation of the SARS-CoV-2 genome, providing a deeper understanding of the evolution of the virus, its proximal origin and revealing patterns of its global spread [16, 17]. These efforts are largely centered on reverse transcription of the viral RNA genome followed by PCR amplicon and hybrid capture based sequencing using Oxford Nanopore and Illumina sequencers [18]. The sequences generated are largely deposited in data repositories such as the sequence read archive (SRA) and GISAID [19].…”