Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination.

BACKGROUND: Between November 2021 and February 2022, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. METHODS: We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. FINDINGS: We identified 18 co-infections, one of which displayed evidence of a low Delta-Omicron recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In the three cases, the 5' end of the viral genome was from the Delta genome and the 3' end from Omicron, including the majority of the spike protein gene, though the breakpoints were different. CONCLUSIONS: Delta-Omicron recombinant viruses were rare, and there is currently no evidence that Delta-Omicron recombinant viruses are more transmissible between hosts compared with the circulating Omicron lineages. FUNDING: This research was supported by the NIH RADx initiative and by the Centers for Disease Control Contract 75D30121C12730 (Helix).

A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping.

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.