Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of Severe Acute Respiratory Syndrome Coronavirus 2 Genomes.

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

Variants of Concern Are Overrepresented Among Postvaccination Breakthrough Infections of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Washington State.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOCs) and had a median Ct of 20.2 (IQR, 17.1-23.3). When compared with 5174 contemporaneous samples sequenced in our laboratory, VOCs were significantly enriched among breakthrough infections (P < .05).

Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis.

PURPOSE: Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. METHODS: In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. RESULTS: ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. CONCLUSION: We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.

Deep sequencing of the human TCRγ and TCRß repertoires suggests that TCRß rearranges after αß and γδ T cell commitment.

T lymphocytes respond to a broad array of pathogens with the combinatorial diversity of the T cell receptor (TCR). This adaptive response is possible because of the unique structure of the TCR, which is composed of two chains, either αß or γδ, that undergo genetic rearrangement in the thymus. αß and γδ T cells are functionally distinct within the host but are derived from a common multipotent precursor. The canonical model for T cell lineage commitment assumes that the γ, δ, and ß chains rearrange before αß or γδ T cell commitment. To test the standard model in humans, we used high-throughput sequencing to catalog millions of TCRγ and TCRß chains from peripheral blood αß and γδ T cells from three unrelated individuals. Almost all sampled αß and γδ T cells had rearranged TCRγ sequences. Although sampled αß T cells had a diverse repertoire of rearranged TCRß chains, less than 4% of γδ T cells in peripheral blood had a rearranged TCRß chain. Our data suggest that TCRγ rearranges in all T lymphocytes, consistent with TCRγ rearranging before T cell lineage commitment. However, rearrangement of the TCRß locus appears to be restricted after T cell precursors commit to the αß T cell lineage. Indeed, in T cell leukemias and lymphomas, TCRγ is almost always rearranged and TCRß is only rearranged in a subset of cancers. Because high-throughput sequencing of TCRs is translated into the clinic for monitoring minimal residual for leukemia/lymphoma, our data suggest the sequencing target should be TCRγ.

Integrating host genomics with surveillance for invasive bacterial diseases.

We tested the feasibility of linking Active Bacterial Core surveillance, a prospective, population-based surveillance system for invasive bacterial disease, to a newborn dried blood spot (nDBS) repository. Using nDBS specimens, we resequenced CD46, putative host gene receptor for Neisseria meningitidis, and identified variants associated with susceptibility to this disease.

LPA and PLG sequence variation and kringle IV-2 copy number in two populations.

BACKGROUND/AIMS: Lp(a) levels have long been recognized as a potential risk factor for coronary heart disease that is almost completely under genetic control. Much of the genetics impacting Lp(a) levels has been attributed to the highly polymorphic LPA kringle IV-2 copy number variant, and most of the variance in Lp(a) levels in populations of European-descent is inversely correlated with kringle IV copy number. However, less of the variance is explained in African-descent populations for the same structural variation. African-descent populations have, on average, higher levels of Lp(a), suggesting other genetic factors contribute to Lp(a) level variability across populations. METHODS: To identify potential cis-acting factors, we re-sequenced the gene LPA for single nucleotide polymorphism (SNP) discovery in 23 European-Americans and 24 African-Americans. We also re- sequenced the neighboring gene plasminogen (PLG) and genotyped the kringle IV copy number variant in the same reference samples. RESULTS: These data are the most comprehensive description of sequence variation in LPA and its relationship with the kringle IV copy number variant. With these data, we demonstrate that only a fraction of LPA sequence diversity has been previously documented. Also, we identify several high frequency SNPs present in the African-American sample but absent in the European-American sample. Finally, we show that SNPs within PLG are not in linkage disequilibrium with SNPs in LPA, and we show that kringle IV copy number variation is not in linkage disequilibrium with either LPA or PLG SNPs. CONCLUSIONS: Together, these data suggest that LPA SNPs could independently contribute to Lp(a) levels in the general population.

The environmental genome project: reference polymorphisms for drug metabolism genes and genome-wide association studies.

The Environmental Genome Project (EGP) has generated a comprehensive resource of commonly occurring single nucleotide polymorphisms (SNPs) in more than six hundred environmental response genes, including a number of drug metabolism genes. The gene-oriented sequence variation discovery carried out by the EGP is complementary to the HapMap and enables genome-wide association studies (GWAS) that survey a large portion of the known common variation. For GWAS focused on drug metabolism genes and phenotypes, it is important to know the proportion of common SNPs covered by the commercially available high-throughput genotyping chips. Herein, we review a subset of Phase I cytochrome P450 genes studied by the EGP, approaches to GWAS, and the sensitivity of available genotyping platforms to capture common sequence variation in this subset of drug metabolism genes.

Linkage disequilibrium in wild mice.

Crosses between laboratory strains of mice provide a powerful way of detecting quantitative trait loci for complex traits related to human disease. Hundreds of these loci have been detected, but only a small number of the underlying causative genes have been identified. The main difficulty is the extensive linkage disequilibrium (LD) in intercross progeny and the slow process of fine-scale mapping by traditional methods. Recently, new approaches have been introduced, such as association studies with inbred lines and multigenerational crosses. These approaches are very useful for interval reduction, but generally do not provide single-gene resolution because of strong LD extending over one to several megabases. Here, we investigate the genetic structure of a natural population of mice in Arizona to determine its suitability for fine-scale LD mapping and association studies. There are three main findings: (1) Arizona mice have a high level of genetic variation, which includes a large fraction of the sequence variation present in classical strains of laboratory mice; (2) they show clear evidence of local inbreeding but appear to lack stable population structure across the study area; and (3) LD decays with distance at a rate similar to human populations, which is considerably more rapid than in laboratory populations of mice. Strong associations in Arizona mice are limited primarily to markers less than 100 kb apart, which provides the possibility of fine-scale association mapping at the level of one or a few genes. Although other considerations, such as sample size requirements and marker discovery, are serious issues in the implementation of association studies, the genetic variation and LD results indicate that wild mice could provide a useful tool for identifying genes that cause variation in complex traits.

Allelic spectrum of the natural variation in CRP.

With the recent completion of the International HapMap Project, many tools are in hand for genetic association studies seeking to test the common variant/common disease hypothesis. In contrast, very few tools and resources are in place for genotype-phenotype studies hypothesizing that rare variation has a large impact on the phenotype of interest. To create these tools for rare variant/common disease studies, much interest is being generated towards investing in re-sequencing either large sample sizes of random chromosomes or smaller sample sizes of patients with extreme phenotypes. As a case study for rare variant discovery in random chromosomes, we have re-sequenced approximately 1,000 chromosomes representing diverse populations for the gene C-reactive protein (CRP). CRP is an important gene in the fields of cardiovascular and inflammation genetics, and its size (approximately 2 kb) makes it particularly amenable medical or deep re-sequencing. With these data, we explore several issues related to the present-day candidate gene association study including the benefits of complete SNP discovery, the effects of tagSNP selection across diverse populations, and completeness of dbSNP for CRP. Also, we show that while deep re-sequencing uncovers potentially medically relevant coding SNPs, these SNPs are fleetingly rare when genotyped in a population-based survey of 7,000 Americans (NHANES III). Collectively, these data suggest that several different types re-sequencing and genotyping approaches may be required to fully understand the complete spectrum of alleles that impact human phenotypes.

Sequence diversity, natural selection and linkage disequilibrium in the human T cell receptor alpha/delta locus.

T cell receptors (TR), through their interaction with the major histocompatibility complex, play a central role in immune responsiveness and potentially immune-related disorders. We resequenced all 57 variable (V) genes in the human T cell receptor alpha and delta (TRA/TRD) locus in 40 individuals of Northern European, Mexican, African-American and Chinese descent. Two hundred and eighty-four single nucleotide polymorphisms (SNPs) were identified. The distribution of SNPs between V genes was heterogeneous, with an average of five SNPs per gene and a range of zero to 15. We describe the patterns of linkage disequilibrium for these newly discovered SNPs and compare these patterns with other emerging large-scale datasets (e.g. Perlegen and HapMap projects) to place our findings into a framework for future analysis of genotype-phenotype associations across this locus. Furthermore, we explore signatures of natural selection across V genes. We find evidence of strong directional selection at this locus as evidenced by unusually high values of Fst.

Genomic regions exhibiting positive selection identified from dense genotype data.

The allele frequency spectrum of polymorphisms in DNA sequences can be used to test for signatures of natural selection that depart from the expected frequency spectrum under the neutral theory. We observed a significant (P = 0.001) correlation between the Tajima's D test statistic in full resequencing data and Tajima's D in a dense, genome-wide data set of genotyped polymorphisms for a set of 179 genes. Based on this, we used a sliding window analysis of Tajima's D across the human genome to identify regions putatively subject to strong, recent, selective sweeps. This survey identified seven Contiguous Regions of Tajima's D Reduction (CRTRs) in an African-descent population (AD), 23 in a European-descent population (ED), and 29 in a Chinese-descent population (XD). Only four CRTRs overlapped between populations: three between ED and XD and one between AD and ED. Full resequencing of eight genes within six CRTRs demonstrated frequency spectra inconsistent with neutral expectations for at least one gene within each CRTR. Identification of the functional polymorphism (and/or haplotype) responsible for the selective sweeps within each CRTR may provide interesting insights into the strongest selective pressures experienced by the human genome over recent evolutionary history.

Comprehensive identification and characterization of diallelic insertion-deletion polymorphisms in 330 human candidate genes.

Despite being the second most frequent type of polymorphism in the genome, diallelic insertion-deletion polymorphisms (indels) have received far less attention in the study of sequence variation. In this report, we describe an approach that can detect indels in the heterozygous state and can comprehensively identify indels in the target sequence. Using this approach, we identified 2393 indels in a set of 330 candidate genes, i.e. an average of seven indels per gene with about two indels per gene being common (minor allele frequency >or=0.1). We compared the population genetic characteristics of indels with substitutions in this data. Our data supported the findings that deletions occur more frequently in the human genome. 5'-UTR and coding regions of the genes showed a significantly lower diversity for indels compared with other regions, suggesting differences in effects of selection on indels and substitutions. Sequence diversity and pairwise linkage disequilibrium (LD) findings of the different populations were similar to earlier results and included a greater skew towards low-frequency variants and a faster rate of LD decay in the African-descent population compared with the non-African populations. Within populations, the allele frequency spectra and LD-decay profiles for indels were similar to substitutions. Overall, the findings suggest that, although the mechanisms giving rise to indels may be different from those causing substitutions, the evolutionary histories of indels and substitutions are similar, and that indels can play a valuable role in association studies and marker selection strategies.

Pattern of sequence variation across 213 environmental response genes.

To promote the clinical and epidemiological studies that improve our understanding of human genetic susceptibility to environmental exposure, the Environmental Genome Project (EGP) has scanned 213 environmental response genes involved in DNA repair, cell cycle regulation, apoptosis, and metabolism for single nucleotide polymorphisms (SNPs). Many of these genes have been implicated by loss-of-function mutations associated with severe diseases attributable to decreased protection of genomic integrity. Therefore, the hypothesis for these studies is that individuals with functionally significant polymorphisms within these genes may be particularly susceptible to genotoxic environmental agents. On average, 20.4 kb of baseline genomic sequence or 86% of each gene, including a substantial amount of introns, all exons, and 1.3 kb upstream and downstream, were scanned for variations in the 90 samples of the Polymorphism Discovery Resource panel. The average nucleotide diversity across the 4.2 MB of these 213 genes is 6.7 x 10(-4), or one SNP every 1500 bp, when two random chromosomes are compared. The average candidate environmental response gene contains 26 PHASE inferred haplotypes, 34 common SNPs, 6.2 coding SNPs (cSNPs), and 2.5 nonsynonymous cSNPs. SIFT and Polyphen analysis of 541 nonsynonymous cSNPs identified 57 potentially deleterious SNPs. An additional eight polymorphisms predict altered protein translation. Because these genes represent 1% of all known human genes, extrapolation from these data predicts the total genomic set of cSNPs, nonsynonymous cSNPs, and potentially deleterious nonsynonymous cSNPs. The implications for the use of these data in direct and indirect association studies of environmentally induced diseases are discussed.

Sequence variation in the human T-cell receptor loci.

Identifying common sequence variations known as single nucleotide polymorphisms (SNPs) in human populations is one of the current objectives of the human genome project. Nearly 3 million SNPs have been identified. Analysis of the relative allele frequency of these markers in human populations and the genetic associations between these markers, known as linkage disequilibrium, is now underway to generate a high-density genetic map. Because of the central role T cells play in immune reactivity, the T-cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system (e.g., asthma, atopy and autoimmunity). Over the past two decades, hundreds of SNPs in the TCR loci have been identified. Most studies have focused on defining SNPs in the variable gene segments which are involved in antigenic recognition. On average, the coding sequence of each TCR variable gene segment contains two SNPs, with many more found in the 5', 3' and intronic sequences of these segments. Therefore, a potentially large repertoire of functional variants exists in these loci. Association between SNPs (linkage disequilibrium) extends approximately 30 kb in the TCR loci, although a few larger regions of disequilibrium have been identified. Therefore, the SNPs found in one variable gene segment may or may not be associated with SNPs in other surrounding variable gene segments. This suggests that meaningful association studies in the TCR loci will require the analysis and typing of large marker sets to fully evaluate the role of TCR loci in common disease susceptibility in human populations.

Historical Relationships Between Research and Resource Management in the Apalachicola River Estuary.

A continuous field research effort has been carried out in the Apalachicola River estuary since March 1972. The information generated from this interdisciplinary study has been directly applied to the management of the Apalachicola resource by means of close associations among local, state, and federal officials and university scientists. During the early years, scientific data were instrumental in the prevention of the impoundment of the Apalachicola River. A series of regional studies was carried out to evaluate various forms of effects due to forestry activities, pesticides, and stormwater runoff from urban areas. A review was made of fisheries problems associated with dredging, overfishing, and marine pollution. Results of such studies were directly applied to local management questions. Research that linked the river wetlands with the estuary, in terms of the input of fresh water, nutrients, and organic matter, served as the basis for the purchase of extensive bottomland tracts. Other initiatives were carried out that were designed to protect the naturally high productivity of the river estuary. Further purchases of estuarine wetlands and barrier island properties were made that formed an almost continuous buffer of publicly held lands between upland developments and critical habitats and important populations of the bay system. A regional management plan was adopted that was designed to limit local municipal development in the estuarine region. Analyses of the long-term scientific data indicated that dominant, commercially important estuarine populations are associated with river flow, local salinity characteristics, and biological (predation, competition) interactions with the salinity regime and food web structure. Such interactions are not straight forward, however; they reflect complex interactions of the freshwater influxes and biological response in the estuary that are not well understood. Species-specific responses to the principal driving factors further complicate the biological relationships of the Apalachicola system. The management of a river-dominated estuary should be based on protection and control of freshwater sources, nutrients, and organic matter with a minimization of physical alterations that often lead to increased salinity stratification and the associated loss of the nursery function of the estuary. There is growing evidence that changes in upland characteristics and within-system habitat alterations are associated with changes in nutrient distributions and salinity relationships and that such changes can have serious impacts on estuarine systems. Issues involving basic changes in estuarine productivity and associated food webs are far more important than those involving species diversity in the management of such resources. Processes such as nutrient flow and salinity alteration underlie the very basis of estuarine productivity and usefulness. Over the past decade, there has been a gradual reduction in the importance of research as an integral part of the planning and management process in the Apalachicola system. This regional trend follows a national pattern. Dredging effects on the river and bay have gone on without effective challenge even though such activities are damaging productive habitats of the system. The once influential Apalachicola oyster fishery remains in disarray following a series of natural disasters and poor management practices. There is a growing problem with the deliberate obstruction of the generation and use of scientific data to determine management policies by various state and federal agencies. The substitution of public relations activities for the development of needed scientific information concerning factors such as the importance of sustained freshwater input to estuaries is further evidence that even the most elaborate and well-conceived management plans can be reversed by political manipulations and short-sighted bureaucratic policies.