A Novel SARS-CoV-2 Viral Sequence Bioinformatic Pipeline Has Found Genetic Evidence That the Viral 3′ Untranslated Region (UTR) Is Evolving and Generating Increased Viral Diversity

Abstract

An unprecedented amount of SARS-CoV-2 sequencing has been performed, however, novel bioinformatic tools to cope with and process these large datasets is needed. Here, we have devised a bioinformatic pipeline that inputs SARS-CoV-2 genome sequencing in FASTA/FASTQ format and outputs a single Variant Calling Format file that can be processed to obtain variant annotations and perform downstream population genetic testing. As proof of concept, we have analyzed over 229,000 SARS-CoV-2 viral sequences up until November 30, 2020. We have identified over 39,000 variants worldwide with increased polymorphisms, spanning the ORF3a gene as well as the 3′ untranslated (UTR) regions, specifically in the conserved stem loop region of SARS-CoV-2 which is accumulating greater observed viral diversity relative to chance variation. Our analysis pipeline has also discovered the existence of SARS-CoV-2 hypermutation with low frequency (less than in 2% of genomes) likely arising through host immune responses and not due to sequencing errors. Among annotated non-sense variants with a population frequency over 1%, recurrent inactivation of the ORF8 gene was found. This was found to be present in the newly identified B.1.1.7 SARS-CoV-2 lineage that originated in the United Kingdom. Almost all VOC-containing genomes possess one stop codon in ORF8 gene (Q27∗), however, 13% of these genomes also contains another stop codon (K68∗), suggesting that ORF8 loss does not interfere with SARS-CoV-2 spread and may play a role in its increased virulence. We have developed this computational pipeline to assist researchers in the rapid analysis and characterization of SARS-CoV-2 variation.

The highly conserved stem-loop II motif is important for the lifecycle of astroviruses but dispensable for SARS-CoV-2

“…We downloaded SARS-CoV-2 complete genome sequences from NCBI which were deposited before November, 2021 as a single FASTA file. The FASTA dataset was processed with a published SARS-CoV-2- freebayes pipeline to call all the variants along the genome 51 . In brief, the single SARS-CoV-2 complete genome FASTA file was decomposed into individual FASTA genome sequences.…”

Coding-Complete Genome Sequences of Alpha and Delta SARS-CoV-2 Variants from Kamphaeng Phet Province, Thailand, from May to July 2021

“…Two nonsense mutations, i.e., G254* and K68* in open reading frame 3a (ORF3a) and ORF8 genes, respectively, were not found in the first alpha variant virus in Thailand but were found in all alpha variant viruses in this study. K68* was reported previously ( 15 ). G254* in ORF3a resulted in the predicted absence of 18 amino acid residues (positions 254 to 271) at the C terminus of the protein, located in a region thought to carry several B cell epitopes ( 16 ).…”

Computational Mapping of the Human-SARS-CoV-2 Protein-RNA Interactome

“…However, we observe a greater variability of RBP binding within shared genomic regions across coronaviruses, for instance in the 5’ and 3’ untranslated regions (UTRs). Viral UTRs are known to play an important role in both pro- and anti-viral responses and recent evidence suggests that evolution of the 3’ UTR is contributing to increased viral diversity (15). Indeed, the 3’ UTR of SARS-CoV-2 shows a severe truncation when compared to SARS-CoV-1 and MERS.…”