Overcoming variant mutation-related impacts on viral sequencing and detection methodologies

Abstract

Prompt and accurate pathogen identification, by diagnostics and sequencing, is an effective tool for tracking and potentially curbing pathogen spread. Targeted detection and amplification of viral genomes depends on annealing complementary oligonucleotides to genomic DNA or cDNA. However, genomic mutations that occur during viral evolution may perturb annealing, which can result in incomplete sequence coverage of the genome and/or false negative diagnostic test results. Herein, we demonstrate how to assess, test, and optimize sequencing and detection methodologies to attenuate the negative impact of mutations on genome targeting efficiency. This evaluation was conducted using in vitro-transcribed (IVT) RNA as well as RNA extracted from clinical SARS-CoV-2 variant samples, including the heavily mutated Omicron variant. Using SARS-CoV-2 as a current example, these results demonstrate how to maintain reliable targeted pathogen sequencing and how to evaluate detection methodologies as new variants emerge.

The Omicron variant BQ.1* with mutations at positions 28,311 and 28,312 in the SARS-CoV-2 N gene have minimal impact on CDC N1 target detection

“…The Primer Monitor tool (https://primermonitor.neb.com/) plays an extremely important role in automatically monitoring the mutational spectrum across a wide range of publicly submitted amplification primer sets. It has proved highly successful in calling out concerning mutations, including revealing the original mutation in Omicron (BA.1) at position 28,311, which overlapped with the CDC 2019-nCoV_N1 target sequence for detection of SARS-CoV-2 in realtime RT-PCR 6 . Some mutations have been found to decrease assay sensitivity (e.g., CDC N2 on G29179U) while others have had no observable impact on sensitivity (e.g., CDC N1 on C28311U or C28311U/A28330G) 1,3,6,7 .…”

“…g ., CDC N2 on G29179U) while others have had no observable impact on sensitivity ( e . g ., CDC N1 on C28311U or C28311U/A28330G) 1,3,6,7 .…”

“…The impact of the 28,311/28,312 C to U mutation on the CDC 2019-nCov_N1 target detection was conducted as previously described 6 . Briefly, a 7-log dilution series (10 7 -10 copies/reaction) was prepared for both the wild-type and the mutant RNA, with 10 ng of Jurkat total RNA (BioChain, R1255815-50) included as an internal control.…”

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

“…Since the PTP analysed here concluded in 2021, several laboratory and bioinformatic developments have occurred, reflecting the evolving nature of the emergent pandemic response. The ARTIC primer scheme was redesigned in terms of laboratory methods and commercial schemes were offered to account for increasing variation (30,31). From a bioinformatic perspective, AusTrakka increased its minimum genome coverage criteria from >50% to >=90% (ACGT bases), which was the basis for the internal quality criterium of several participants.…”

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance

“…Since the PTP analysed here concluded in 2021, several laboratory and bioinformatic developments have occurred, reflecting the evolving nature of the emergent pandemic response. The ARTIC primer scheme was redesigned in terms of laboratory methods and commercial schemes were offered to account for increasing variation [30,31]. From a bioinformatic perspective, AusTrakka increased its minimum genome coverage criteria from >50 % to >=90 % (ACGT bases), which was the basis for the internal quality criteria of several participants.…”