Whole-genome Next-generation Sequencing Research Articles

Long-term wastewater monitoring of SARS-CoV-2 viral loads and variants at the major international passenger hub Amsterdam Schiphol Airport: A valuable addition to COVID-19 surveillance

Wastewater-based epidemiological surveillance at municipal wastewater treatment plants has proven to play an important role in COVID-19 surveillance. Considering international passenger hubs contribute extensively to global transmission of viruses, wastewater surveillance at this type of location may be of added value as well. The aim of this study is to explore the potential of long-term wastewater surveillance at a large passenger hub as an additional tool for public health surveillance during different stages of a pandemic. Here, we present an analysis of SARS-CoV-2 viral loads in airport wastewater by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) from the beginning of the COVID-19 pandemic in Feb 2020, and an analysis of SARS-CoV-2 variants by whole-genome next-generation sequencing from Sep 2020, both until Sep 2022, in the Netherlands. Results are contextualized using (inter)national measures and data sources such as passenger numbers, clinical surveillance data and national wastewater surveillance data. Our findings show that wastewater surveillance was possible throughout the study period, irrespective of measures, as viral loads were detected and quantified in 98.6 % (273/277) of samples. Emergence of SARS-CoV-2 variants, identified in 91.0 % (161/177) of sequenced samples, coincided with increases in viral loads. Furthermore, trends in viral load and variant detection in airport wastewater closely followed, and in some cases preceded, trends in national daily average viral load in wastewater and variants detected in clinical surveillance. Wastewater-based epidemiology at a large international airport is a valuable addition to classical COVID-19 surveillance and the developed expertise can be applied in pandemic preparedness plans for other (emerging) pathogens in the future.

Transcriptional profile features in patients with early and late preeclampsia

Aim: to assess the molecular mechanisms in developing various clinical phenotypes of preeclampsia (PE) by analyzing specific placental tissue transcriptome patterns. Materials and Methods. The prospective observational comparative study in parallel groups enrolled 43 pregnant women divided into 2 groups: main group – 23 pregnant women with diagnosed PE and control group – 20 apparently healthy women with uncomplicated pregnancy course, delivery and the postpartum period. To examine PE phenotypic features, the main group of pregnant women with PE was subsequently divided into 2 subgroups according to the date of pathology onset: early (n = 10) and late (n = 13) PE. Using the whole-genome next-generation sequencing (NGS), a comparative analysis of altered 18 microRNA level in placental tissue was carried out. Results. Pregnant women with early PE compared to the control group were characterized by significantly low expression level for hsa-miR-656-3p (p < 0.001), hsa-miR-323a-5p (p = 0.017), hsa-miR-519c-3p (p = 0.019), hsa-let-7i-5p (p = 0.019), hsa-miR-433-3p (p = 0.019), hsa-let-7g-5p (p = 0.030), hsa-miR-214-5p (p = 0.030), hsa-miR-27a-5p (p = 0.031), hsa-miR-339-5p (p = 0.041), hsa-miR-524-5p (p = 0.045), hsa-miR-1283 (p = 0.049) and high expression for hsa-miR-151a-5p (p = 0.007), hsa-miR-4521 (p = 0.018), hsa-miR-30d-5p (p = 0.026), hsa-miR-548l (p = 0.027), hsa-miR-133b (p = 0.034), hsa-miR-424-5p (p = 0.042), hsa-miR-211-5p (p = 0.049). Patients with late PE had significantly decreased expression for hsa-miR-656-3p (p = 0.050) andhsa-miR-574-3p (p = 0.017) as well as a significantly higher for hsa-miR-211-5p (p = 0.001) compared to the control group. Subgroup of women with early vs. late onset PE was characterized by significantly decreased expression level for hsa-miR-323-5p (p = 0.007) and overexpressed hsa-miR-30d-5p (p = 0.002), hsa-miR-5481 (p = 0.027). Conclusion. The noted multidirectional expression for some microRNAs in subgroups of PE patients confirms the validity for stratification of such pathology based on two distinct phenotypic manifestations (early and late forms) and indicates the existence of different pathophysiological vectors in PE formation.

The complete chloroplast genome of the halophyte flowering plant Suaeda monoica from Jeddah, Saudi Arabia.

The complete chloroplast genome (plastome) of the annual flowering halophyte herb Suaeda monoica Forssk. ex J. F. Gmel. family (Amaranthaceae) that grows in Jeddah, Saudi Arabia, was identified for the first time in this study. Suaeda monoica is a medicinal plant species whose taxonomic classification remains controversial. Further, studying the species is useful for current conservation and management efforts. In the current study, the full chloroplast genome S. monoica was reassembled using whole-genome next-generation sequencing and compared with the previously published chloroplast genomes of Suaeda species. The chloroplast genome size of Suaeda monoica was 151,789bp, with a single large copy of 83,404bp, a small single copy of 18,007bp and two inverted repeats regions of 25,189bp. GC content in the whole genome was 36.4%. The cp genome included 87 genes that coded for proteins, 37 genes coding for tRNA, 8 genes coding for rRNA and one non-coding pseudogene. Five chloroplast genome features were compared between S. monoica and S. japonica, S. glauca, S. salsa, S. malacosperma and S. physophora. Among Suaeda genus and equal to most angiosperms chloroplast genomes, the RSCU values were conservative. Two pseudogenes (accD and ycf1), rpl16 intron and ndhF-rpl32 intergenic spacer, were highlighted as suitable DNA barcodes for different Suaeda species. Phylogenetic analyses show Suaeda cluster into three main groups; one in which S. monoica was closer to S. salsa. The obtained result provided valuable information on the characteristics of the S. monoica chloroplast genome and the phylogenetic relationships.

SARS-COV-2 GENOMIC SURVEILLANCE IN BULGARIA INDICATES DIVERSE DYNAMICS DRIVEN BY MULTIPLE INTRODUCTIONS OF DIFFERENT VIRAL VARIANTS IN 2022

Background. Evolution of the emerging SARS-CoV-2 variants raises concerns about the possibility of accelerated transmission, disease severity, diagnostic challenges, and reduced vaccine effectiveness in the ever-evolving COVID-19 pandemic worldwide. Objectives for this study were to build a comprehensive national system for monitoring and genomic surveillance of SARS-CoV-2 and to identify the introduced virus variants in the country. Methods. We analyzed SARS-CoV-2 infections in 7948 representative clinical samples collected in medical institutions in different geographical regions of the country in 2022. Whole-genome next-generation sequencing of SARS-CoV-2 was performed on samples from randomly selected SARS-CoV-2-positive individuals by using a modified ARTIC v3-tailed amplicon method. A bioinformatic and phylogenetic analyses of the obtained sequences was carried out. Results. Significant dynamics was observed in the spread of viral variants in 2022, which is characterized by the introduction and spread of multiple SARS-CoV-2 variants. The phylogenomic analysis identified a high genetic heterogeneiety composed of a total of 152 different viral clades divided into 3 main supergroups: 114 (75.0%) of which were Omicron sub-variants, 35 (23.0%) Delta sub-variants, and 3 (2.0%) recombinant forms. Conclusion. Viral variants and their sub-clades with different potentials to impact disease severity were identified and the information was immediately published for use by decision-makers and the scientific community. The global pandemic of COVID-19 has shown the importance of molecular biological surveillance, which is an indispensable element of the modern approach in the fight against infectious diseases.

Genomic and Temporal Analysis of Deletions Correlated to qRT-PCR Dropout in N Gene in Alpha, Delta and Omicron Variants.

Since the first SARS-CoV-2 outbreak, mutations such as single nucleotide polymorphisms (SNPs) and insertion/deletions (INDELs) have changed and characterized the viral genome sequence, structure and protein folding leading to the onset of new variants. The presence of those alterations challenges not only the clinical field but also the diagnostic demand due to failures in gene detection or incompleteness of polymerase chain reaction (PCR) results. In particular, the analysis of understudied genes such as N and the investigation through whole-genome next generation sequencing (WG-NGS) of regions more prone to mutate can help in the identification of new or reacquired mutations, with the aim of designing robust and long-lasting primers. In 48 samples of SARS-CoV-2 (including Alpha, Delta and Omicron variants), a lack of N gene amplification was observed in the genomes analyzed through WG-NGS. Three gene regions were detected hosting the highest number of SNPs and INDELs. In several cases, the latter can interfere deeply with both the sensitivity of diagnostic methodologies and the final protein folding. The monitoring over time of the viral evolution and the reacquisition among different variants of the same mutations or different alterations within the same genomic positions can be relevant to avoid unnecessary consumption of resources.

West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022.

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations.

The complete chloroplast genome of Rhododendron shanii W.P. Fang (Ericaceae), a endemic plant from the Southern Dabie Mountains of China

Rhododendron shanii W.P. Fang 1983 (Ericaceae) is woody plant naturally distributed in the southwest of Anhui, China. The complete chloroplast genome sequence of R. shanii was generated by whole-genome next-generation sequencing data and assembled based on three Rhododendron species chloroplast genome. The complete chloroplast genome sequence of R. shanii was 204,170 bp and divided into four distinct regions: small single-copy region (2615 bp), large single-copy region (107,189 bp), and a pair of inverted repeat regions (47,183 bp). The genome annotation displayed 150 genes, including 95 protein-coding genes, 47 tRNA genes, and eight rRNA genes. Phylogenetic analysis with the Ericaceae reported chloroplast genomes revealed that R. shanii is sister to the clade comprising R. delavayi, R. griersonianum and R. platypodum.

Evaluation of cfDNA as an early detection assay for dense tissue breast cancer

A cell-free DNA (cfDNA) assay would be a promising approach to early cancer diagnosis, especially for patients with dense tissues. Consistent cfDNA signatures have been observed for many carcinogens. Recently, investigations of cfDNA as a reliable early detection bioassay have presented a powerful opportunity for detecting dense tissue screening complications early. We performed a prospective study to evaluate the potential of characterizing cfDNA as a central element in the early detection of dense tissue breast cancer (BC). Plasma samples were collected from 32 consenting subjects with dense tissue and positive mammograms, 20 with positive biopsies and 12 with negative biopsies. After screening and before biopsy, cfDNA was extracted, and whole-genome next-generation sequencing (NGS) was performed on all samples. Copy number alteration (CNA) and single nucleotide polymorphism (SNP)/insertion/deletion (Indel) analyses were performed to characterize cfDNA. In the positive-positive subjects (cases), a total of 5 CNAs overlapped with 5 previously reported BC-related oncogenes (KSR2, MAP2K4, MSI2, CANT1 and MSI2). In addition, 1 SNP was detected in KMT2C, a BC oncogene, and 9 others were detected in or near 10 genes (SERAC1, DAGLB, MACF1, NVL, FBXW4, FANK1, KCTD4, CAVIN1; ATP6V0A1 and ZBTB20-AS1) previously associated with non-BC cancers. For the positive–negative subjects (screening), 3 CNAs were detected in BC genes (ACVR2A, CUL3 and PIK3R1), and 5 SNPs were identified in 6 non-BC cancer genes (SNIP1, TBC1D10B, PANK1, PRKCA and RUNX2; SUPT3H). This study presents evidence of the potential of using cfDNA somatic variants as dense tissue BC biomarkers from a noninvasive liquid bioassay for early cancer detection.

Retrieving Good-Quality Salmonella Genomes From the GenBank Database Using a Python Tool, SalmoDEST.

With the advent of next-generation whole-genome sequencing (WGS), the need for good-quality and well-characterised Salmonella genomes has increased over the past years. Good-quality complete genomes are often required for assembly reference mapping or phylogenetic single nucleotide polymorphism (SNP) analysis. Complete genomes or contigs from specific sources or serovars are also searched for clustering analysis or source attribution studies. Therefore, new bioinformatics tools are needed for the extraction of good-quality and well-characterised genomes from public databases. Here, we developed SalmoDEST, an open-source Python tool capable of extracting Salmonella genomes with a coverage higher than 50x and genome length over 4Mb from the GenBank database in the form of complete genomes or contigs, with verification of the serovar to which they belong and identification of the corresponding multi locus sequence type (MLST) profile. To validate the ability to SalmoDEST to screen for and retrieve genomes of good quality, we compared our results for S. Typhi complete genome with those available in the literature and extracted Salmonella genomes from bovine sources strains isolated worldwide. Finally, we provide in this study a list of 239 complete genomes for 123 serovars of Salmonella of high quality. SalmoDEST is a handy and easy-to-use open-source tool to extract complete genomes or contigs that can be routinely used in public health, food safety and research laboratories. SalmoDEST (SALMOnella Download gEnome Serotype sT) is available at https://github.com/I-Guy/SalmoDEST.

CSF MicroRNAs Reveal Impairment of Angiogenesis and Autophagy in Parkinson Disease.

Background and ObjectivesWe assessed longitudinal changes in CSF microRNAs (miRNAs) in patients with moderately severe Parkinson disease.MethodsWe used next-generation whole-genome miRNA sequencing to determine CSF miRNA expression in 75 patients with Parkinson disease after single random ascending doses of nilotinib and longitudinal miRNA expression after daily nilotinib, 150 and 300 mg, vs placebo for 1 year.ResultsSignificant changes in the expression of miRNAs that control genes and pathways that regulate angiogenesis, autophagy, and the blood-brain-barrier components, primarily collagen, were observed over 1 year, suggesting impairment of these pathways in Parkinson progression in these patients. Different miRNAs that indicate activation of genes associated with autophagy flux and clearance and angiogenesis were significantly altered in the nilotinib, 300 mg vs 150 mg, or placebo group, and these changes correlated with clinical outcomes. No changes were observed in miRNAs after a single dose of nilotinib vs placebo.DiscussionThis study suggests vascular and autophagy defects in Parkinson progression. Nilotinib, 300 mg, reverses these effects via alteration of miRNA expression, suggesting epigenomic changes that may underlie long-term disease-modifying effects.Trial Registration InformationClinical trial registration number: NCT02954978.

Primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement: a case report and literature review

BackgroundWe present a unique case of primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement in a young Chinese female patient.Case presentationThe patient presented with a mass in her right breast that rapidly increased in size over approximately 2 months. Upper gastrointestinal endoscopy showed a giant serpentine ulcer in the stomach. Biopsy was performed, and microscopic inspection revealed that the fibrous tissue was diffusely involved by medium to large abnormal lymphocytes. The cytoplasm was low to moderate. The tumor cells had irregular nuclei and inconspicuous nucleoli. The lymphoid cells were strongly immunoreactive to CD20, CD3, CD4, CD56, TIA-1, EBER, and Ki-67 (90%). Epstein-Barr virus genomes were also found in tumor cells by in situ hybridization. A whole-body positron emission tomography (PET)-CT scan revealed intense FDG uptake in the right breast and greater curvature of the stomach. Monoclonal rearrangements of the T cell receptor (TCR-γ) and immunoglobulin heavy chain (IgH) were identified by genetic analysis. Whole-genome next-generation sequencing was performed, and up to 12 gene mutations, including a frameshift mutation in exon 4 of the BCOR (G97Rfs*87; 44.3%) gene and a base substitution mutation (Q61H 17.6%) in exon 3 of the KRAS gene, were detected. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the database for annotation, visualization, and integrated discovery, which showed that rare primary breast CD20-positive extranodal NK/T cell lymphoma had a unique genetic background compared with diffuse large B cell lymphoma and extranodal NK/T cell lymphoma without CD20 expression. The patient received four cycles of the modified SMILE regimen. The second whole-body PET-CT scan revealed that the right breast mass was significantly smaller than before; additionally, FDG uptake in the stomach wall disappeared.ConclusionsSystemic examination, extensive immunohistochemistry, and molecular profiling are essential for an accurate diagnosis. More similar cases are required to clarify the biological pathways and even the potential molecular mechanisms of rare lymphomas, which may help direct further treatment.

Assessing hepatitis C virus distribution among vulnerable populations in London using whole genome sequencing: results from the TB-REACH study

Background: Injecting drugs substantially increases the risk of hepatitis C virus (HCV) infection and is common in vulnerable population groups, such as the homeless and prisoners. Capturing accurate data on relative genotype distribution within these groups is essential to inform strategies to reduce HCV transmission. The aim of this study was to utilise a next-generation whole-genome sequencing method recently validated by Public Health England, in order to produce near complete HCV genomes. Methods: In total, 98 HCV positive patients were recruited from homeless hostels and drug treatment services through the National Health Services (NHS) Find and Treat (F&T) Service between May 2011 and June 2013 in London, UK. Samples were sequenced by Next-generation sequencing, with 88 complete HCV genomes constructed by a de novo assembly pipeline. They were analysed phylogenetically for an estimate of their genetic distance. Results: Of the 88 complete HCV genomes, 50/88 (56.8%) were genotype 1; 32/88 (36.4%) genotype 3; 4/88 (4.5%) genotype 2; and 1/88 (1.1%) for genotypes 4 and 6 each. Subtype 1a had the highest number of samples (51.1%), followed by subtype 3a (35.2%), 1b (5.7%), and 2b (3.4%). Samples collected from drug treatment services had the highest number of genotype 1 (69%); genotypes 4 and 6 were only found from samples collected in homeless shelters. Small clusters of highly related genomic sequences were observed both across and within the vulnerable groups sampled. Conclusions: Subsequent phylogenetic analysis provides a first indication that there are related HCV sequences amongst the three vulnerable population groups, reflecting their overlapping social behaviours. This study is the first presentation of whole genome HCV sequences from such vulnerable groups in London and paves the way for similar research in the future.

High throughput diagnostics and dynamic risk assessment of SARS-CoV-2 variants of concern.

BackgroundThe rise of new SARS-CoV-2 variants worldwide requires global molecular surveillance strategies to support public health control. Early detection and evaluation of their associated risk of spreading within the population are pivotal.MethodsBetween April 2020 and February 2021, the UK Lighthouse Labs Network at Alderley Park tested more than eight million nose and throat swab samples for the presence of SARS-CoV-2, via PCR. The assay targeted three genomic regions of the virus: N, Orf1ab and S. Whole-genome next-generation sequencing was used to confirm positive PCR results. Positive results were mapped using the postal district origin of samples to allow real-time tracking of the spread of a new variant through the UK.FindingsIn mid-November 2020, the assay identified an increasing number of S gene negative, N and Orf1ab positive samples. Whole-genome sequencing demonstrated that the loss of S gene detection was due to the appearance of a SARS-CoV-2 lineage (B.1.1.7) designated as Variant of concern (VOC) 202012/01. By the beginning of January 2021, the new SARS-CoV-2 VOC comprised 70% of daily positive samples tested at Alderley Park and ∼98% by the end of February 2021.InterpretationThe timeline view identified the rapid spread of the new SARS-CoV-2 variant across England during the first three weeks of December. Coupling high-throughput diagnostics and molecular surveillance was pivotal to the early detection of the spread of this variant. The availability of real-time tracking of an emerging variant is an important new tool to inform decision-making authorities for risk mitigation. In a respiratory pandemic, a tool for the timely response to the emergence and spread of a novel variant is vital, even more so when a variant is associated with the enhanced transmission, as has occurred with VOC 202012/01.

The complete chloroplast genome sequence of Bambusa stenoaurita (Bambusoideae)

Bambusa stenoaurita is an excellent sympodial bamboo species, which is cultivated for its shoots in some parts of China. Here, we sequenced and reported the complete chloroplast genome of B. stenoaurita for the first time. The complete chloroplast genome sequence of B. stenoaurita was generated by de novo assembly using whole-genome next-generation sequencing. The genome was 139,451 bp in total length, including a large single-copy region of 82,958 bp, a small single-copy region of 12,897 bp, a pair of invert repeats regions of 21,798 bp. The plastid genome contained 134 genes including 87 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 23 chloroplast genomes demonstrates that B. stenoaurita is closely related to B. emeiensis in Bambusoideae.

Noncanonical outcomes of break-induced replication produce complex, extremely long-tract gene conversion events in yeast.

Long-tract gene conversions (LTGC) can result from the repair of collapsed replication forks, and several mechanisms have been proposed to explain how the repair process produces this outcome. We studied LTGC events produced from repair collapsed forks at yeast fragile site FS2. Our analysis included chromosome sizing by contour-clamped homogeneous electric field electrophoresis, next-generation whole-genome sequencing, and Sanger sequencing across repair event junctions. We compared the sequence and structure of LTGC events in our cells to the expected qualities of LTGC events generated by proposed mechanisms. Our evidence indicates that some LTGC events arise from half-crossover during BIR, some LTGC events arise from gap repair, and some LTGC events can be explained by either gap repair or “late” template switch during BIR. Also based on our data, we propose that models of collapsed replication forks be revised to show not a one-end double-strand break (DSB), but rather a two-end DSB in which the ends are separated in time and subject to gap repair.

Human Papillomavirus Detection by Whole-Genome Next-Generation Sequencing: Importance of Validation and Quality Assurance Procedures.

Next-generation sequencing (NGS) yields powerful opportunities for studying human papillomavirus (HPV) genomics for applications in epidemiology, public health, and clinical diagnostics. HPV genotypes, variants, and point mutations can be investigated in clinical materials and described in previously unprecedented detail. However, both the NGS laboratory analysis and bioinformatical approach require numerous steps and checks to ensure robust interpretation of results. Here, we provide a step-by-step review of recommendations for validation and quality assurance procedures of each step in the typical NGS workflow, with a focus on whole-genome sequencing approaches. The use of directed pilots and protocols to ensure optimization of sequencing data yield, followed by curated bioinformatical procedures, is particularly emphasized. Finally, the storage and sharing of data sets are discussed. The development of international standards for quality assurance should be a goal for the HPV NGS community, similar to what has been developed for other areas of sequencing efforts including microbiology and molecular pathology. We thus propose that it is time for NGS to be included in the global efforts on quality assurance and improvement of HPV-based testing and diagnostics.

NPSV: A simulation-driven approach to genotyping structural variants in whole-genome sequencing data

BackgroundStructural variants (SVs) play a causal role in numerous diseases but are difficult to detect and accurately genotype (determine zygosity) in whole-genome next-generation sequencing data. SV genotypers that assume that the aligned sequencing data uniformly reflect the underlying SV or use existing SV call sets as training data can only partially account for variant and sample-specific biases.ResultsWe introduce NPSV, a machine learning–based approach for genotyping previously discovered SVs that uses next-generation sequencing simulation to model the combined effects of the genomic region, sequencer, and alignment pipeline on the observed SV evidence. We evaluate NPSV alongside existing SV genotypers on multiple benchmark call sets. We show that NPSV consistently achieves or exceeds state-of-the-art genotyping accuracy across SV call sets, samples, and variant types. NPSV can specifically identify putative de novo SVs in a trio context and is robust to offset SV breakpoints.ConclusionsGrowing SV databases and the increasing availability of SV calls from long-read sequencing make stand-alone genotyping of previously identified SVs an increasingly important component of genome analyses. By treating potential biases as a “black box” that can be simulated, NPSV provides a framework for accurately genotyping a broad range of SVs in both targeted and genome-scale applications.

Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross‐breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole‐genome next‐generation sequencing (NGS) in autopolyploid crops. To apply the NGS‐based bulked segregant method, QTL‐seq was modified. (1) Single parent‐specific simplex (unique for one homologous chromosome) single‐nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL‐seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase PCR (RT-qPCR) to detect three nonsynonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2-positive specimens from our San Francisco Bay Area population. Between 1 December 2020 and 1 March 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K plus N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing of a validation subset of 229 specimens and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed the rapid emergence of the L452R variant in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Evaluation of tools for identifying large copy number variations from ultra-low-coverage whole-genome sequencing data

BackgroundDetection of copy number variations (CNVs) from high-throughput next-generation whole-genome sequencing (WGS) data has become a widely used research method during the recent years. However, only a little is known about the applicability of the developed algorithms to ultra-low-coverage (0.0005–0.8×) data that is used in various research and clinical applications, such as digital karyotyping and single-cell CNV detection.ResultHere, the performance of six popular read-depth based CNV detection algorithms (BIC-seq2, Canvas, CNVnator, FREEC, HMMcopy, and QDNAseq) was studied using ultra-low-coverage WGS data. Real-world array- and karyotyping kit-based validation were used as a benchmark in the evaluation. Additionally, ultra-low-coverage WGS data was simulated to investigate the ability of the algorithms to identify CNVs in the sex chromosomes and the theoretical minimum coverage at which these tools can accurately function. Our results suggest that while all the methods were able to detect large CNVs, many methods were susceptible to producing false positives when smaller CNVs (< 2 Mbp) were detected. There was also significant variability in their ability to identify CNVs in the sex chromosomes. Overall, BIC-seq2 was found to be the best method in terms of statistical performance. However, its significant drawback was by far the slowest runtime among the methods (> 3 h) compared with FREEC (~ 3 min), which we considered the second-best method.ConclusionsOur comparative analysis demonstrates that CNV detection from ultra-low-coverage WGS data can be a highly accurate method for the detection of large copy number variations when their length is in millions of base pairs. These findings facilitate applications that utilize ultra-low-coverage CNV detection.