Dataset Information

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.

ABSTRACT: Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.

SUBMITTER: Brandt D

PROVIDER: S-EPMC8518987 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.

Brandt David D Simunovic Marina M Busche Tobias T Haak Markus M Belmann Peter P Jünemann Sebastian S Schulz Tizian T Klages Levin Joe LJ Vinke Svenja S Beckstette Michael M Pohl Ehmke E Scherer Christiane C Sczyrba Alexander A Kalinowski Jörn J

Viruses 20210918 9

Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 ...[more]

PMID: 34578452

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Project description:To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans.IMPORTANCE During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.

Project description:The development and innovation of next generation sequencing (NGS) and the subsequent analysis tools have gain popularity in scientific researches and clinical diagnostic applications. Hence, a systematic comparison of the sequencing platforms and variant calling pipelines could provide significant guidance to NGS-based scientific and clinical genomics. In this study, we compared the performance, concordance and operating efficiency of 27 combinations of sequencing platforms and variant calling pipelines, testing three variant calling pipelines-Genome Analysis Tool Kit HaplotypeCaller, Strelka2 and Samtools-Varscan2 for nine data sets for the NA12878 genome sequenced by different platforms including BGISEQ500, MGISEQ2000, HiSeq4000, NovaSeq and HiSeq Xten. For the variants calling performance of 12 combinations in WES datasets, all combinations displayed good performance in calling SNPs, with their F-scores entirely higher than 0.96, and their performance in calling INDELs varies from 0.75 to 0.91. And all 15 combinations in WGS datasets also manifested good performance, with F-scores in calling SNPs were entirely higher than 0.975 and their performance in calling INDELs varies from 0.71 to 0.93. All of these combinations manifested high concordance in variant identification, while the divergence of variants identification in WGS datasets were larger than that in WES datasets. We also down-sampled the original WES and WGS datasets at a series of gradient coverage across multiple platforms, then the variants calling period consumed by the three pipelines at each coverage were counted, respectively. For the GIAB datasets on both BGI and Illumina platforms, Strelka2 manifested its ultra-performance in detecting accuracy and processing efficiency compared with other two pipelines on each sequencing platform, which was recommended in the further promotion and application of next generation sequencing technology. The results of our researches will provide useful and comprehensive guidelines for personal or organizational researchers in reliable and consistent variants identification.

Dataset Information

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.

Publications

Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines.

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