A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture

Abstract

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

High Throughput Sequencing for the Detection and Characterization of RNA Viruses

“…It was determined that amplicon sequencing had greater on target reads, though CBPH demonstrated a significantly higher standard deviation of genome coverage a more accurate depiction of SNV s (Samorodnitsky et al, 2015 ). Furthermore, Nasir et al ( 2020 ) noted that CBPH provided an advantage over amplification based protocols such as tiling amplicon approaches due to the absence of amplification artifacts.…”

Multiplex Sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

“…The exponential growth of the publicly available SARS-CoV-2 genome sequences attributable to the rapid genome sequencing, development of data analysis work ow, and data sharing by researchers worldwide [11][12][13]. Currently, most of the sequencing work ows were created for the use of the Oxford Nanopore and Illumina's sequencing platforms [16][17][18]. The ARTIC protocol is one of the most widely used sequencing methods using the Oxford Nanopore platform [11].…”

Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

“…In consequence, Fluidigm amplification based whole genome sequencing has limitations in sequencing low titer samples. For RNA samples with 1000 GE/μl or lower concentration, multiplex RT-PCR based methods or SARS-CoV-2 hybridization-based enrichment method might be a more suitable choice for whole genome sequencing (26–28). The addition of a first-strand cDNA synthesis step prior to Fluidigm amplification, with a change of Fluidigm thermocycling program from RT-PCR to PCR, may help increase genome coverage for low titer samples.…”