Whole Genome Sequencing Methods Research Articles

GenoVi, an open-source automated circular genome visualizer for bacteria and archaea.

The increase in microbial sequenced genomes from pure cultures and metagenomic samples reflects the current attainability of whole-genome and shotgun sequencing methods. However, software for genome visualization still lacks automation, integration of different analyses, and customizable options for non-experienced users. In this study, we introduce GenoVi, a Python command-line tool able to create custom circular genome representations for the analysis and visualization of microbial genomes and sequence elements. It is designed to work with complete or draft genomes, featuring customizable options including 25 different built-in color palettes (including 5 color-blind safe palettes), text formatting options, and automatic scaling for complete genomes or sequence elements with more than one replicon/sequence. Using a Genbank format file as the input file or multiple files within a directory, GenoVi (i) visualizes genomic features from the GenBank annotation file, (ii) integrates a Cluster of Orthologs Group (COG) categories analysis using DeepNOG, (iii) automatically scales the visualization of each replicon of complete genomes or multiple sequence elements, (iv) and generates COG histograms, COG frequency heatmaps and output tables including general stats of each replicon or contig processed. GenoVi's potential was assessed by analyzing single and multiple genomes of Bacteria and Archaea. Paraburkholderia genomes were analyzed to obtain a fast classification of replicons in large multipartite genomes. GenoVi works as an easy-to-use command-line tool and provides customizable options to automatically generate genomic maps for scientific publications, educational resources, and outreach activities. GenoVi is freely available and can be downloaded from https://github.com/robotoD/GenoVi.

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested. This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Quinolone-resistant Escherichia coli at the interface between humans, poultry and their shared environment- a potential public health risk

BackgroundCommensal Escherichia coli residing in the guts of humans and animals are reservoirs of multidrug resistance (MDR) genes, including quinolone resistance genes, in humans and poultry. This study aimed to characterize quinolones resistance in E. coli recovered from poultry workers, chickens, and poultry farm/market environments in Abuja, Nigeria.MethodsThis was a cross-sectional study conducted between December 2018 and April 2019 comprising poultry workers, chickens and their poultry farm/market environments. This study characterized E. coli isolates from stool, faecal and environmental samples using antimicrobial susceptibility testing and whole-genome sequencing methods. Core-genome multilocus sequences-based phylogeny was used to determine the relatedness between quinolone-resistant E. coli isolates. Data were analyzed using descriptive statistics.ResultsOf 110 E. coli isolates, quinolone-resistant phenotypes were observed in 68.2% (n = 75) isolates. Whole-genome sequencing detected plasmid-mediated quinolone resistance (PMQR) genes in 63.6% (n = 70) isolates. The most prevalent PMQR gene detected in 56 of these 70 E. coli isolates was qnrS1, followed by qnrB19 in 14 isolates and aac(6’)-lb-cr in two isolates. Fifteen ciprofloxacin and 19 nalidixic acid-resistant isolates respectively showed double mutations in the quinolone-resistance determining regions (QRDRs) of gyrA, with single or double mutations in parC, and a single mutation in parE. The most prevalent amino-acid substitutions observed were S83L + D87N in gyrA (46.5%, n = 20), S80I in parC (51.2%, n = 22) and S458A in parE (14%, n = 6). About 2.9% (2/70) of PMQR isolates were extended-spectrum beta-lactamase (ESBL) producers while 2.9% (2/70) had plasmid-mediated colistin resistance (PMCR) genes.ConclusionsPMQR genes were prevalent in E. coli isolates recovered from healthy humans, chickens and poultry farm/market environments. PMCR genes (mcr-1.1) occurred in PMQR-positive isolates recovered from manure and drinking water originating from poultry farm/market environments. It was found that the gene encoding ESBL coexisted with qnrS-positive isolates of human and avian origin. Horizontal transfer of PMQR genes among E. coli isolates in the human-poultry-environment interface has public health implications for the spread of antimicrobial resistance. Relevant government agencies should enforce regulations to restrict the use of critically important antimicrobials in poultry production.

LINE1-Mediated Reverse Transcription and Genomic Integration of SARS-CoV-2 mRNA Detected in Virus-Infected but Not in Viral mRNA-Transfected Cells.

SARS-CoV-2 sequences can be reverse-transcribed and integrated into the genomes of virus-infected cells by a LINE1-mediated retrotransposition mechanism. Whole-genome sequencing (WGS) methods detected retrotransposed SARS-CoV-2 subgenomic sequences in virus-infected cells overexpressing LINE1, while an enrichment method (TagMap) identified retrotranspositions in cells that did not overexpress LINE1. LINE1 overexpression increased retrotranspositions about 1000-fold as compared to non-overexpressing cells. Nanopore WGS can directly recover retrotransposed viral and flanking host sequences, but its sensitivity depends on the depth of sequencing (a typical 20-fold sequencing depth would only examine 10 diploid cell equivalents). In contrast, TagMap enriches the host-virus junctions and can interrogate up to 20,000 cells and is able to detect rare viral retrotranspositions in LINE1 non-overexpressing cells. Although Nanopore WGS is 10-20-fold more sensitive per tested cell, TagMap can interrogate 1000-2000-fold more cells and, therefore, can identify infrequent retrotranspositions. When comparing SARS-CoV-2 infection and viral nucleocapsid mRNA transfection by TagMap, retrotransposed SARS-CoV-2 sequences were only detected in infected but not in transfected cells. Retrotransposition in virus-infected cells, in contrast to transfected cells, may be facilitated because virus infection, in contrast to viral RNA transfection, results in significantly higher viral RNA levels and stimulates LINE1 expression by causing cellular stress.

Diagnosis of Dengue Virus Infections Imported to Hungary and Phylogenetic Analysis of Virus Isolates

Dengue virus is one of the most important arbovirus infections of public health concern. Between 2017 and June 2022, 75 imported dengue infections were confirmed by laboratory diagnostic methods in Hungary. Our study aimed to isolate the imported Dengue strains and characterize them by whole-genome sequencing. Laboratory diagnosis of imported infections was carried out using both serological and molecular methods. Virus isolation was attempted on Vero E6 cell lines. An in-house amplicon-based whole-genome sequencing method was applied for the detailed molecular characterization of the isolated virus strains. From 75 confirmed Dengue infected patients, 68 samples were used for virus isolation. Isolation and whole-genome sequencing were successful in the case of eleven specimens. Isolated strains belonged to Dengue-1,-2,-3 serotypes. The isolated strains corresponded to the circulating genotypes of the visited geographic area, and some of the genotypes were linked with more severe DENV cases in the literature. We found that multiple factors, including viral load, specimen type, and patient antibody status, influence the isolation efficacy. Analysis of imported DENV strains can help estimate the outcomes of a possible local DENV transmission in Hungary, a threat from the near future.

Genetic and Phenotypic Characterization of the First Canadian Case of Ambler Class A Carbapenemase FRI-8

This study investigated the mechanism of carbapenem resistance in an Enterobacter cloacae complex positive by the modified carbapenem inactivation method (mCIM) but negative by the Rosco Neo-Rapid Carb Kit, β CARBA, and conventional PCR for common carbapenemase genes (KPC, NDM, OXA-48, IMP, VIM, GES, and IMI/NMC). Using whole genome sequencing (WGS) data we confirmed the identification of Enterobacter asburiae (ST1639) and the presence of blaFRI-8 located on a 148kb IncFII(Yp) plasmid. This is the first occurrence of a clinical isolate harboring the FRI-8 carbapenemase and the second occurrence of FRI in Canada. This study highlights the need to use both WGS and phenotypic screening methods for detection of carbapenemase-producing strains if we consider the growing diversity of carbapenemases.

WHOLE-GENOME SINGLE MOLECULE REAL-TIME SEQUENCING OF SARS-CoV-2 OMICRON.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harbouring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p<0.001) on our dataset, and the median genome coverage with the target capture protocol (98.6% [IQR: 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], (p<0.001)). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p<0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants. This article is protected by copyright. All rights reserved.

Mutational characterization of Omicron SARS-CoV-2 lineages circulating in Chhattisgarh, a central state of India.

The emergence of the Omicron SARS-CoV-2 variant from various states of India in early 2022 has caused fear of its rapid spread. The lack of such reports from Chhattisgarh (CG), a central state in India, has prompted us to identify the Omicron circulating lineages and their mutational dynamics. Whole-genome sequencing (WGS) of SARS-CoV-2 was performed in 108 SARS-CoV-2 positive combined samples of nasopharyngeal and oropharyngeal swabs obtained from an equal number of patients. All 108 SARS-CoV-2 sequences belonged to Omicron of clade 21L (84%), 22B (11%), and 22D (5%). BA.2 and its sub-lineages were predominantly found in 93.5% of patients, BA.5.2 and its sub-lineage BA.5.2.1 in 4.6% of patients, and B.1.1.529 in 2% of patients. Various BA.2 sub-lineages identified were BA.2 (38%), BA.2.38 (32%), BA.2.75 (9.25%), BA.2.56, BA.2.76, and BA.5.2.1 (5% each), BA.2.74 (4.6%), BA.5.2.1 (3.7%), BA.2.43 and B.1.1.529 (1.8% each), and BA.5.2 (0.9%). Maximum mutations were noticed in the spike (46), followed by the nucleocapsid (5), membrane (3), and envelope (2) genes. Mutations detected in the spike gene of different Omicron variants were BA.1.1.529 (32), BA.2 (44), BA.2.38 (37), BA.2.43 (38), BA.2.56 (30), BA.2.74 (31), BA.2.75 (37), BA.2.76 (32), BA.5.2, and BA.5.2.1 (38 similar mutations). The spike gene showed the signature mutations of T19I and V213G in the N-terminal domain (NTD), S373P, S375F, T376A, and D405N in receptor-binding domain (RBD), D614G, H655Y, N679K, and P681H at the furin cleavage site, N764K and D796K in fusion peptide, and Q954H and N969K in heptapeptide repeat sequence (HR)1. Notably, BA.2.43 exhibited a novel mutation of E1202Q in the C terminal. Other sites included ORF1a harboring 13 mutations followed by ORF1b (6), ORF3a (2), and ORF6 and ORF8 (1 mutation each). BA.2 followed by BA.2.38 was the predominant Omicron lineage circulating in Chhattisgarh. BA.2.75 could supersede other Omicron due to its mutational consortium advantage. The periodical genomic monitoring of Omicron variants is thus required for real-time assessment of circulating strains and their mutational-induced severity.

Genetic Characteristics of Lipooligosaccharide and Capsular Polysaccharide of Campylobacter jejuni from Different Sources in China

ObjectiveTo determine the distribution of two important virulence factors [lipooligosaccharide (LOS) and capsular polysaccharide (CPS)] in Campylobacter jejuni (C. jejuni) isolated from different sources in China and to develop a rapid screening method for Guillain–Barré syndrome (GBS)-associated strains. MethodsWhole-genome sequencing was carried out for 494 C. jejuni strains. The OrthoMCL software was used to define the LOS/CPS gene clusters. CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software. Real-time Polymerase chain reaction (PCR) was developed with the unique sequences of specific CPS types. ResultsNine novel and 29 previously confirmed LOS classes were identified. LOS classes A, B, and C were the most common (48.2%, 238/494) among the 494 strains. Twenty-six capsular types were identified in 448 strains. HS2, HS4c, HS5/31, HS19, and HS8/17 were the most frequent CPS genotypes (58.7%, 263/448). Strains of 17 CPS genotypes (strain number > 5) had one or two prevalent LOS classes (P < 0.05). Multiplex real-time PCR for rapid identification of HS2, HS19, and HS41 was developed and validated with strains of known serotypes. ConclusionOur results describe the genetic characteristics of the important virulence factors in C. jejuni strains in China. The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China.

ScONE-seq: A single-cell multi-omics method enables simultaneous dissection of phenotype and genotype heterogeneity from frozen tumors.

Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell whole-genome RNA sequencing (scWGS-RNA-seq) methods demonstrate utility with intact cells from fresh samples. Among them, many are not applicable to frozen samples that cannot produce intact single-cell suspensions. We have developed scONE-seq, a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples. We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects. We identified a unique transcriptionally normal-like tumor clone by analyzing a 2-year frozen astrocytoma sample, demonstrating that performing single-cell multi-omics interrogation on biobanked tissue by scONE-seq could enable previously unidentified discoveries in tumor biology.

137. Comparison of genotypic resistance pattern between Enterococcus faecium and Enterococcus faecalis to better understand the occurrence of mutations.

Abstract Background Enterococcus is one of the major causes of nosocomial infections many with vancomycin resistance. About 50,000 infections are reported per year due to vancomycin-resistant Enterococci. Understanding the genotypic mutations can help recognize the potential burden for antibiotic resistance that exists in a population. In this study, using whole genome sequencing (WGS) we aim to analyze the prevalence of genetic resistance markers for commonly used antibiotics for Enterococcal treatment. Methods Whole genome sequencing was performed using the NextSeq (Illumina Inc., CA) on 60 isolates of E. faecium and 29 isolates of E. faecalis. The isolates were obtained from two different Detroit area hospitals from 2017-2019. The data from WGS was analyzed using EPISEQ CSTM (BIOMÉRIEUX, Marcy l ‘Etoile, France) bioinformatic database to obtain specific resistance genes which would correspond to commonly used antibiotics for treatment of Enterococcal infections. Results Among the 89 Enterococcus faecium and Enterococcus faecalis investigated, we identified a total of 33 unique resistance genes across 18 classes of antibiotics (Table 1). We detected genetic mutations efmA, pbp5, tet(L), liar, liaS unique to E. faecium and only emeA unique to E. faecalis. E. faecium and E. faecalis share 14 similar resistance genes. E. faecium and E. faecalis had 12 and 7 unique resistance genes, respectively but are of same drug classes. The most common antibiotic classes include aminoglycosides, tetracyclines, quinolones, beta-lactams, glycopeptides, macrolides, and pyrimidine analogs. Table 1:Enterococcus faecium and E. faecalis genetic mutations and drug class resistance Conclusion Enterococcus faecium had a greater number of resistance genes with glycopeptide, aminoglycoside, and beta lactam resistance when compared to Enterococcus faecalis. Whereas Enterococcus faecalis had more resistance genes associated with macrolides, tetracyclines, pyrimidine analogs, and Bactrim. In general, both appear to have many genes that encode for resistance to major classes of antibiotics which are used for treatment of Enterococcal infections. Further comparison with phenotypic susceptibility testing data would help better understand the practical implications of our resistance gene testing. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|GlaxoSmithKline plc.: Receiving symposia honoraria|GlaxoSmithKline plc.: GlaxoSmithKline plc.-sponsored study 212502|Merck: Advisor/Consultant|qpex: Advisor/Consultant|Shionogi: Grant/Research Support|Spero: Advisor/Consultant Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

Genetic markers associated with resistance to radioiodine therapy in thyroid cancer patients: Prospective cohort study

Background. The indication for radiotherapy in oncological practice are metastases of differentiated thyroid cancer after thyroidectomy, the presence of distant metastases, or stage N1b, or negative dynamics of blood thyroglobulin levels after thyroidectomy for thyroid cancer. The mechanism of action of radiotherapy is based on provoking double-stranded DNA breaks. It is important to study the role of polymorphisms of NFKB1, ATM, ATG16L2 and ATG10 genes, products of which are involved in the processes of DNA damage response pathway and autophagy, in the formation of resistance to radioiodine therapy of thyroid cancer patients.&#x0D; Aim. To examine the association between NFKB1, ATM, ATG16L2 and ATG10 polymorphisms and resistance to radioiodine therapy in thyroid cancer patients.&#x0D; Materials and methods. The study included 181 patients (37 men, 144 women; mean age 53.515.7 years) with histologically confirmed thyroid cancer and a history of thyroidectomy who received radioiodine therapy. Carriage of single-nucleotide polymorphisms (rs230493) NFKB1, (rs11212570) ATM, (rs10898880) ATG16L2 and (rs10514231, rs1864183, rs4703533) ATG10 was determined by real-time PCR using TaqMan kits.&#x0D; Results. Among 181 patients, resistance to radioiodine therapy was observed in 11 (6.1%) cases. No significant associations between the individual polymorphisms and resistance to radioiodine therapy were obtained, p0.05. Haplotype analysis showed that carriage of the C-C ATG10 rs10514231-rs1864183 haplotype was associated with an increased risk of developing resistance to radioiodine therapy, p=0.04.&#x0D; Conclusion. Further studies on large samples of radioiodine therapy-resistant patients using whole-genome sequencing methods are required to specify the role of genetic factors in the response to 131I therapy.

SARS-CoV-2 Variants Identification; A Fast and Affordable Strategy Based on Partial S-Gene Targeted PCR Sequencing.

A considerable number of new SARS-CoV-2 lineages have emerged since the first COVID-19 cases were reported in Wuhan. As a few variants showed higher COVID-19 disease transmissibility and the ability to escape from immune responses, surveillance became relevant at that time. Single-nucleotide mutation PCR-based protocols were not always specific, and consequently, determination of a high number of informative sites was needed for accurate lineage identification. A detailed in silico analysis of SARS-CoV-2 sequences retrieved from GISAID database revealed the S gene 921 bp-fragment, positions 22784-23705 of SARS-CoV-2 reference genome, as the most informative fragment (30 variable sites) to determine relevant SARS-CoV-2 variants. Consequently, a method consisting of the PCR-amplification of this fragment, followed by Sanger's sequencing and a "single-click" informatic program based on a reference database, was developed and validated. PCR-fragments obtained from clinical SARS-CoV-2 samples were compared with homologous variant-sequences and the resulting phylogenetic tree allowed the identification of Alpha, Delta, Omicron, Beta, Gamma, and other variants. The data analysis procedure was automatized and simplified to the point that it did not require specific technical skills. The method is faster and cheaper than current whole-genome sequencing methods; it is available worldwide, and it may help to enhance efficient surveillance in the fight against the COVID-19 pandemic.

Whole Genome Sequencing for Detection of Prognostically Relevant Genetic Abnormalities in Paediatric Acute Myeloid Leukaemia

Background Genetics is important in the diagnosis of paediatric acute myeloid leukaemia (AML). A number of chromosomal abnormalities and mutations have prognostic significance and timely genetic analysis is essential for risk stratification. The recent Myechild 01 clinical trial for children with AML required testing for >20 different genetic abnormalities to assign newly diagnosed patients into three prognostic genetic risk groups: good, intermediate and poor (Figure A). Throughout the trial a combination of techniques was employed to detect these abnormalities; including chromosomal analysis, FISH for cytogenetically cryptic abnormalities, notably the poor risk fusions: t(5;11)(q35;p15)/NUP98::NSD1, inv(16)(p13q24)/CBFA2T3::GLIS2, t(7;12)(q36;p13)/ETV6::MNX1, as well as molecular testing for NPM1, FLT3-ITD and CEBPA mutations. Here, we present a small retrospective study demonstrating that whole genome sequencing (WGS) has the potential to detect all prognostically significant abnormalities in childhood AML within a single test. Methods WGS was performed on matched diagnostic and remission samples from 25 paediatric AML patients entered to the Myechild 01 treatment trial. Remission samples, minimal residual disease negative by flow cytometry and/or molecular testing, were used as germline controls. For validation, whole transcriptome sequencing (WTS) was performed on diagnostic RNA from each patient in the study. Results Leukaemia associated genetic abnormalities were detected in all 25 patients, including structural variants associated with gene fusions (n=14), single nucleotide variants (SNV) (n=20) and/or copy number abnormalities/aneuploidies (n=17). All gene fusions and mutations were confirmed by WTS. WGS reliably detected all risk stratifying abnormalities identified by standard techniques (Figure B), including 11 patients with gene fusions: RUNX1::RUNX1T1 (n=6), CBFB::MYH11 (n=3), KMT2A::MLLT3 (n=1) and KMT2A::MLLT10 (n=1) and 3 patients with copy number abnormalities: del(5q) (n=2) and del(12p) (n=1) and one patient with an NPM1 mutation. Among 10 patients with no risk stratifying abnormalities detected by standard techniques, WGS identified risk defining genetic features in 3 of them. Two patients could be reclassified as good risk due to the discovery of biallelic CEBPA mutations. Interestingly, one of the CEBPA positive patients presented with a homozygous mutation (VAF=0.97), which WGS revealed to be the consequence of copy number neutral loss of heterozygosity for the long arm of chromosome 19. A single patient could be reclassified as poor risk due to the finding of a rearrangement of MECOM with CDK6. Cytogenetics showed a deletion of 7q as the sole karyotypic abnormality, which was revealed to be an unbalanced der(7)t(3;7)(q21.2;q26.2) translocation by WGS. Over-expression of MECOM was confirmed by WTS. This patient also had a GATA2 mutation. A risk defining abnormality was not detected by either standard testing or WGS in 7 patients, however 5 of them presented with chromosomal abnormalities which were confirmed and further characterised by WGS, including aneuploidies (n=3) and/or structural rearrangements (n=4). Across the cohort, WGS detected a wide range of abnormalities not currently used in the risk stratification algorithm, including the fusions genes: NPM1::MLF1 (n=1) and PICALM::MLLT10 (n=2), which, although not stratifying in Myechild 01, have been reported to be associated with a good and a poor prognosis, respectively. SNV in genes commonly mutated in AML were observed in 19 patients (76%) including NRAS (n=6), KIT (n=3) EZH2 (n=3) and GATA2 (n=2). It has been suggested that the co-occurrence of mutations may be used to refine risk groups in childhood AML, notably the presence of KIT and NRAS mutations in RUNX1::RUNXT1 and CBFB::MYH11 positive leukaemia, respectively. Conclusion This small pilot study has demonstrated the ability of WGS to identify a wide range of genetic abnormalities that are currently used for risk stratification in childhood AML. The full spectrum of genomic events observed here highlights the potential of WGS to refine risk stratification and identify novel genetic changes as therapeutic targets in future trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Simple Cost-Effective Method for Whole-Genome Sequencing, Haplotyping, and Assembly.

In this chapter, we describe single-tube long fragment read (stLFR), a simple preparation method for whole-genome sequencing and physical haplotyping based on the DNA co-barcoding strategy. Similar to LFR, stLFR applies the concept of adding the same barcode to subfragments derived from the same long DNA molecule. However, instead of a 384-well plate, stLFR uses the surface of micron-sized magnetic beads to create millions of virtual compartments in a single reaction tube. This is enabled by a split and pool barcoded bead preparation process capable of generating ~500,000 copies of the same unique barcode, from a library of ~3.6 billion unique barcodes, on each bead. The instruments and devices used in the stLFR process are easily accessible in nearly all standard molecular biology laboratories, and the cost of reagents can be as low as 30 dollars per sample. stLFR libraries can be sequenced by standard second-generation sequencing instruments (e.g., MGI or Illumina devices), and the barcode sharing information enables detection and phasing of all variations, including large structural variations. In addition, stLFR data can be used to scaffold contigs and de novo assemble genomes.

Whole genome sequencing and characteristics of extended-spectrum beta-lactamase producing Escherichia coli isolated from poultry farms in Banaskantha, India.

Worldwide dissemination of extended-spectrum -lactamase (ESBL)-producing Escherichia coli constitutes an emerging global health issue, with animal food products contributing as potential reservoirs. ESBL E. coli infection is associated with the high mortality and mobility rate in developing countries due to less susceptibility to antibiotics. The present study aimed to elucidate the molecular characteristics and sequence-based analysis of ESBL E. coli in the Gujarat state of India. This study included 108 E. coli strains were isolated from different poultry farms (broiler and layer) in the Banaskantha District. PCR was employed to identify genotypic ESBL-producing antimicrobial resistance genes. Overall, a high occurrence of ESBL genes was found in poultry farms due to the high usage of antimicrobials. The PCR analysis revealed that 79.62% of isolates were detected positive with one or more ESBL genes. Among them, blaTEM (63.88%) was found to be the predominant genotype, followed by blaSHV (30.55%) and blaOXA (28.70%). In the blaCTX-M group, a higher occurrence was observed in blaCTX-M-9 (23.14%), followed by blaCTX-M-2 (24.07%) and blaCTX-M-1 (22.22%). We used the whole-genome sequencing (WGS) method to evaluate the antimicrobial resistance genes, virulence factors, single nucleotide polymorphisms (SNPs), plasmid replicons, and plasmid-mediated AMR genes of one ESBL E. coli isolated. We examined the genetic relatedness of a human pathogenic E. coli strain by comparing its sequence with the broad geographical reference E. coli sequences. Escherichia coli ST 681 was determined using multi-locus sequence typing. We compared our findings to the reference sequence of Escherichia coli str. K- 12 substr. MG1655. We found 24,937 SNPs with 21,792 in the genic region, 3,145 in the intergenic region, and six InDels across the genome. The WGS analysis revealed 46 antimicrobial resistance genes and seven plasmid-mediated AMR genes viz., tetA, qnrS1, dfrA14, sul2, aph(3”)-lb, aph(6)-ld, and Aph(3’)-la. The ST 681 was found to have Cib, traT, and terC virulence factors and two plasmid replicons, IncFII(pHN7A8) and IncI1-I(Alpha). This study revealed a higher occurrence of ESBL E. coli detected in poultry.

P484 Expanding VGVI—evidence for distinct Cryptococcus gattii (decagattii) endemic to the American Southwest

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesWe aimed to understand the nature of autochthonous Cryptococcus gattii clinical and veterinary cases identified in Arizona, a state in the American Southwest, a locale well outside of the known endemic regions.MethodsWhole-genome sequencing and phylogenomic comparative analyses were conducted on four unrelated isolates collected from recent cases along with other relevant C. gattii genomes.ResultsPhylogenomic analysis grouped the Arizona genomes with a previously known set of Mexican isolate genomes, labelled as VGVI or C. decagattii. These genomes are clearly delineated from the nearest major molecular type (VGIII), but show no recombination with other molecular types or species of C. gattii.See Figures below.ConclusionThese findings expand VGVI into a definitive clade and establish this molecular type as a clinically important and distinct population. These findings also expand the known Cryptococcus ecological range into a previously unrecognized endemic area, typified by extreme heat and aridity.

A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples.

ABSTRACTDengue virus (DENV) is the most globally prevalent member of the genus Flavivirus in the family Flaviviridae, which can be classified into four serotypes. Historically, molecular epidemiological studies of DENV depended on E gene sequencing. The development of next-generation sequencing (NGS) allowed its application to viral whole-genome sequencing (WGS). In this study, we report the improvement of the existing WGS process for DENV by optimizing the primer design procedure, designing serotype-specific primer panels and reducing the sizes of amplicons. A total of 31 DENV-positive serum samples belonging to 4 serotypes and 9 genotypes of DENV were involved in the validation of the primer panels. The threshold cycle (CT) values of these samples ranged from 23.91 to 35.11. The validation results showed that the length of consensus sequences generated at a coverage depth of 20× or more ranged from 10,370 to 10,672 bp, with 100.00% coverage of the open reading frames and 97.34% to 99.52% coverage of the DENV genome. The amplification efficiency varied across amplicons, genotypes, and serotypes of DENVs. These results indicate that the serotype-specific primer panels allow users to obtain the whole genome of DENV directly from clinical samples, providing a universal, rapid, and effective tool for the integration of genomics with dengue surveillance.IMPORTANCE Dengue virus (DENV) is becoming the most globally prevalent arbovirus. The number of people living under the threat of DENV is increasing year by year. With the development of next-generation sequencing (NGS) technology, whole-genome sequencing (WGS) has been more and more widely used in infectious disease surveillance and molecular epidemiological studies. DENV population sequencing by NGS can increase our understanding of the changing epidemiology and evolution of the DENV genome at the molecular level, which demands universal primer panels and combination with NGS platforms. Multiplex PCR with a short-amplicon approach proved superior for amplifying viral genomes from clinical samples, particularly when the viral RNA was present at low concentrations. Additionally, DENV are known for their genetic diversity within serotype groups and geographical regions, so the primer panels we designed focused on universality, which would be useful in future local DENV outbreaks.

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Community-led risk analysis of direct-to-consumer whole-genome sequencing.

Direct-to-consumer (DTC) genetic testing is cheaper and more accessible than ever before; however, the intention to combine, reuse, and resell this genetic information as powerful data sets is generally hidden from the consumer. This financial gain is creating a competitive DTC market, reducing the price of whole-genome sequencing (WGS) to under 300 USD. Entering this transition from single-nucleotide polymorphism-based DTC testing to WGS DTC testing, individuals looking for access to their whole-genomic information face new privacy and security risks. Differences between WGS and other methods of consumer genetic tests are left unexplored by regulation, leading to the application of legal data anonymization methods on whole-genome data, and questionable consent methods. Large representative genomic data sets are important for research and improve the standard of medicine and personalized care. However, these data can also be used by market players, law enforcement, and governments for surveillance, population analyses, marketing purposes, and discrimination. Here, we present a summary of the state of WGS DTC genetic testing and its current regulation, through a community-based lens to expose dual-use risks in consumer-facing biotechnologies.