Conventional Sequencing Methods Research Articles

Advances in mapping analysis of ribonucleic acid modifications through sequencing

Over 170 chemical modifications have been discovered in various types of ribonucleic acids (RNAs), including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). These RNA modifications play crucial roles in a wide range of biological processes such as gene expression regulation, RNA stability maintenance, and protein translation. RNA modifications represent a new dimension of gene expression regulation known as the "epitranscriptome". The discovery of RNA modifications and the relevant writers, erasers, and readers provides an important basis for studies on the dynamic regulation and physiological functions of RNA modifications. Owing to the development of detection technologies for RNA modifications, studies on RNA epitranscriptomes have progressed to the single-base resolution, multilayer, and full-coverage stage. Transcriptome-wide methods help discover new RNA modification sites and are of great importance for elucidating the molecular regulatory mechanisms of epitranscriptomics, exploring the disease associations of RNA modifications, and understanding their clinical applications. The existing RNA modification sequencing technologies can be categorized according to the pretreatment approach and sequencing principle as direct high-throughput sequencing, antibody-enrichment sequencing, enzyme-assisted sequencing, chemical labeling-assisted sequencing, metabolic labeling sequencing, and nanopore sequencing technologies. These methods, as well as studies on the functions of RNA modifications, have greatly expanded our understanding of epitranscriptomics. In this review, we summarize the recent progress in RNA modification detection technologies, focusing on the basic principles, advantages, and limitations of different methods. Direct high-throughput sequencing methods do not require complex RNA pretreatment and allow for the mapping of RNA modifications using conventional RNA sequencing methods. However, only a few RNA modifications can be analyzed by high-throughput sequencing. Antibody enrichment followed by high-throughput sequencing has emerged as a crucial approach for mapping RNA modifications, significantly advancing the understanding of RNA modifications and their regulatory functions in different species. However, the resolution of antibody-enrichment sequencing is limited to approximately 100-200 bp. Although chemical crosslinking techniques can achieve single-base resolution, these methods are often complex, and the specificity of the antibodies used in these methods has raised concerns. In particular, the issue of off-target binding by the antibodies requires urgent attention. Enzyme-assisted sequencing has improved the accuracy of the localization analysis of RNA modifications and enables stoichiometric detection with single-base resolution. However, the enzymes used in this technique show poor reactivity, specificity, and sequence preference. Chemical labeling sequencing has become a widely used approach for profiling RNA modifications, particularly by altering reverse transcription (RT) signatures such as RT stops, misincorporations, and deletions. Chemical-assisted sequencing provides a sequence-independent RNA modification detection strategy that enables the localization of multiple RNA modifications. Additionally, when combined with the biotin-streptavidin affinity method, low-abundance RNA modifications can be enriched and detected. Nevertheless, the specificity of many chemical reactions remains problematic, and the development of specific reaction probes for particular modifications should continue in the future to achieve the precise localization of RNA modifications. As an indirect localization method, metabolic labeling sequencing specifically localizes the sites at which modifying enzymes act, which is of great significance in the study of RNA modification functions. However, this method is limited by the intracellular labeling of RNA and cannot be applied to biological samples such as clinical tissues and blood samples. Nanopore sequencing is a direct RNA-sequencing method that does not require RT or the polymerase chain reaction (PCR). However, challenges in analyzing the data obtained from nanopore sequencing, such as the high rate of false positives, must be resolved. Discussing sequencing analysis methods for various types of RNA modifications is instructive for the future development of novel RNA modification mapping technologies, and will aid studies on the functions of RNA modifications across the entire transcriptome.

Abstract 2928: Solving the cancer mutation conundrum: A single cell, massively parallel approach for cancer mutation discovery, genome modelling and functional characterization

Abstract Large scale genomic studies are cataloguing tens of thousands of cancer mutations across a wide spectrum of tumor types. Conventional DNA sequencing methods have limitations in terms of identify mutations at high resolution among single cells. Moreover, determining cancer mutations’ phenotype and function remains an enormous challenge - most mutations are assigned to the category of variants of unknown significance. In-silico functional predictions of cancer mutations are frequently used as a stopgap solution. However, these computational methods lack the ability to make refined biological and functional predictions. We demonstrated a new single cell experimental strategy that accelerates the discovery of cancer mutations and their characterization. This genomic technology framework does the following: (1) identify disease-related mutations at the resolution of individual cells from tumors or cell lines; (2) genome engineer these candidate mutations into cellular experimental systems for further biological characterization. First, we developed a single cell approach to identify cancer somatic mutations and even gene fusion rearrangements found in coding regions of mRNAs. Primary tumors are processed into single cell RNA-seq libraries and analyzed with nanopore long read sequencing. Across thousands of cells from primary tumors, we identified single cell mutations from the nanopore long reads that covered cancer gene transcripts. This approach allows us to even identify cancer rearrangements at single cell resolution. Next, we used short-read single cell transcriptomes to characterize the cell type and determine the gene expression characteristics of those cells with mutations. For single cell phenotypic studies, we developed a highly multiplexed technology using CRISPR base editors to directly engineer hundreds of mutations into their original genes among single cells and determine their transcriptional phenotype. Among specific individual cells, nanopore-based long-read sequencing identifies these modelled mutations directly from a target’s transcript sequence. Then, we integrate this single cell mutation data with the short-read transcriptome profile from the same individual cell. Therefore, we determine both the genotype and phenotype of a given engineered mutation from an individual cell. In summary, our work demonstrates a new, direct and highly scalable method for identifying, modelling and functionally assessing cancer mutation phenotype at single cell resolution. This approach enables the broader assessment of cancer mutations impact on tumor biology and clinical ramifications. Citation Format: Susan M. Grimes, Heonseok Kim, Sharmili Roy, Anuja Sathe, Billy T. Lau, Hanlee P. Ji. Solving the cancer mutation conundrum: A single cell, massively parallel approach for cancer mutation discovery, genome modelling and functional characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2928.

First molecular characterization of Blastocystis subtypes from domestic animals (sheep and cattle) and their animal-keepers in Ilam, western Iran: A zoonotic concern.

A total of 360 fecal samples were randomly collected from 150 cattle, 150 sheep, and 60 humans (30 people with close animal contact and 30 individuals without close animal contact) at 10 farms in Ilam, western Iran from June 2022 to August 2023. All samples were directly examined for Blastocystis by zinc sulfate flotation, followed by microscopic observation. Positive samples were further subtyped using conventional PCR and sequencing methods. A mean prevalence of 5.3% (16/300) was estimated for Blastocystis infection among examined animals, with 6% and 4.7% for cattle and sheep, respectively. Among the people who had close and non-close animal contact, 16.7% (5/30) and 3.3% (1/30) were infected with Blastocystis, respectively (p < 0.05). All 22 positive samples were successfully sequenced at the SSU rRNA locus. Accordingly, Blastocystis isolates infecting domestic animals in Ilam belonged to the four STs (ST1-ST3, and ST10). Of the 16 animal isolates, nine sequences (four ST10, three ST3, and two ST1) were related to cattle, and seven sequences (three ST10, two ST3, and two ST2) were isolated from sheep. Among the six human isolates, ST3 was the most predominant ST, followed by STs 1, 2, 6, and 7 (one case each). Of note, ST1-ST3 were isolated in various farms both from animals and their breeders, which indicates the possible circulation of these STs between animal and human populations.

A hand-powered SERS-microfluidic chip for circulating tumor DNA detection from whole blood

Portable devices for rapid, automated and quantitative nucleic acid detection from whole blood is highly desirable for point-of-care (POC) molecular diagnostics. Conventional sequencing and polymerase chain reaction methods usually require trained personnel to deal with the time-consuming procedures, from sample purification to target amplification. To promote POC disease diagnosis and monitoring, we herein developed a hand-powered microfluidic chip for circulating tumor DNA (ctDNA) analysis from whole blood. On the one hand, the chip allowed rapid blood cell removal through a microfluidic filter trench. On the other hand, the ultrasensitivity of surface enhanced Raman spectroscopy (SERS) technique enabled amplification-free DNA mutation screening. Meanwhile, a finger-driven vacuum system was incorporated into the system, which achieved microfluidic pumping without external pumps, tubes or power sources. The developed SERS chip achieved fast screening of EGFR E746-A750 mutation in 35 min, reaching a limit of detection down to 100 fM in blood. Due to the simplicity, fast analysis, low cost and quantitative readout, the chip is expected to expand the availability of molecular diagnosis into resource-limited and bedside settings.

High-throughput identification and quantification of bacterial cells in the microbiota based on 16S rRNA sequencing with single-base accuracy using BarBIQ.

Bacteria often function as a community, called the microbiota, consisting of many different bacterial species. The accurate identification of bacterial types and the simultaneous quantification of the cells of each bacterial type will advance our understanding of microbiota; however, this cannot be performed by conventional 16S rRNA sequencing methods as they only identify and quantify genes, which do not always represent cells. Here, we present a protocol for our developed method, barcoding bacteria for identification and quantification (BarBIQ). In BarBIQ, the 16S rRNA genes of single bacterial cells are amplified and attached to a unique cellular barcode in a droplet. Sequencing the tandemly linked cellular barcodes and 16S rRNA genes from many droplets (representing many cells with unique cellular barcodes) and clustering the sequences using the barcodes determines both the bacterial type for each cell based on 16S rRNA gene and the number of cells for each bacterial type based on the quantity of barcode types sequenced. Single-base accuracy for 16S rRNA sequencing is achieved via the barcodes and by avoiding chimera formation from 16S rRNA genes of different bacteria using droplets. For data processing, an easy-to-use bioinformatic pipeline is available ( https://github.com/Shiroguchi-Lab/BarBIQ_Pipeline_V1_2_0 ). This protocol allows researchers with experience in molecular biology but without bioinformatics experience to perform the process in ~2 weeks. We show the application of BarBIQ in mouse gut microbiota analysis as an example; however, this method is also applicable to other microbiota samples, including those from the mouth and skin, marine environments, soil and plants, as well as those from other terrestrial environments.

Phylogenetic and network analysis of Pediculus humanus in Nigeria reveal the presences of clade E body lice and novel haplotypes.

An investigation was conducted for the first time to determine the prevalence and genetic diversity of human lice, for the first time in Nigeria, using conventional PCR and sequencing methods. Three mitochondrial genes, cytochrome oxidase subunit 1 (cox1), cytochrome b (cytb), and 12S rRNA of Nigerian human lice, were amplified, sequenced, and analyzed. Overall, high prevalence (72.5%; 103/142) of lice infestation was recorded among the examined volunteers. Head lice infestation was more common 63 (61.2%) than body lice infestation 34 (33.0%). Co-infestation with both head and body lice was recorded in six humans (5.8%). The Nigerian human lice specimens were placed mostly into clade A with few in clade E, including body lice for the first time. Six, three, and eight haplotypes of Nigerian human lice were obtained for the cytb, cox1, and 12S rRNA genes, respectively. Additionally, one (E51), three (A31, A32, and E5), and six (A20, A21, A23, A24, A30, and E1) novel haplotypes were recorded for cox1, cytb, and 12S rRNA, respectively, from the Nigerian specimens which were corroborated by the ML phylogenetic trees and MJ network analyses. Genetic diversity indices indicate minimal variation in the parameters analyzed among the clades of the three genes. However, a statistically significant Snn test, negative Tajima's D test for clade A (cox1 and 12S rRNA genes), and negative Fu and Li's D test in clade A for cox1 gene indicate a geographical structure and the signature of population expansion of the Nigerian human lice. The findings from this study provide additional data on the human lice structure in Africa.

Fluorometric Detection of SARS-CoV-2 Single-Nucleotide Variant L452R Using Ligation-Based Isothermal Gene Amplification.

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant was first discovered, several variants showing different infectivity and immune responses have emerged globally. As the conventional method, whole-genome sequencing following polymerase chain reaction (PCR) is currently used for diagnosis of SARS-CoV-2 mutations. However, these conventional PCR-based direct DNA sequencing methods are time-consuming, complicated, and require expensive DNA sequencing modules. Here, we developed a fluorometric method for the accurate detection of a single missense mutation of U to G in the spike (S) gene that changes leucine to arginine (L452R) in SARS-CoV-2 genomic RNA. Our method for the detection of single-nucleotide mutations (SNM) in the viral RNA genome includes RNA sequence-dependent DNA ligation and tandem isothermal gene amplification methods, such as strand displacement amplification (SDA) and rolling circle amplification (RCA) generating G-quadruplex (GQ). In the presence of SNM in the viral RNA, ligation of both ends of the probe DNAs occurs between 5'-phosphorylated hairpin DNA and linear probe DNA that can discriminate a single base mismatch. The ligated DNAs were then extended to generate long-stem hairpin DNAs that are subjected to the first isothermal gene amplification (SDA). SDA produces multitudes of short ssDNA from the long-stem hairpin DNAs, which then serve as primers by annealing to circular padlock DNA for the second isothermal gene amplification (RCA). RCA produces a long stretch of ssDNA containing GQ structures. Thioflavin T (ThT) is then intercalated into GQ and emits green fluorescence, which allows the fluorometric identification of SARS-CoV-2 variants. This fluorometric analysis sensitively distinguished SNM in the L452R variant of SARS-CoV-2 RNA as low as 10 pM within 2 h. Hence, this fluorometric detection method using ligation-assisted tandem isothermal gene amplification can be applied for the diagnosis of SARS-CoV-2 SNM variants with high accuracy and sensitivity, without the need for cumbersome whole-genome DNA sequencing.

Identification of carrier status of Xp22.31 microdeletions associated with X-linked ichthyosis at the single-cell level using haplotype linkage analysis by karyomapping.

Currently, owing to the limitations of high-throughput sequencing depth and the allele dropout caused by the whole-genome amplification, detection of chromosomal variants in embryos with CNVs <5 Mb is unsatisfactory at the single-cell level using only conventional sequencing methods. Therefore, here we aimed to use a strategy of preimplantation genetic testing for monogenic (PGT-M) to compensate for the shortcomings of conventional sequencing methods. The purpose of this study is to report the effectiveness of haplotype linkage analysis by karyomapping for preimplantation diagnosis microdeletion diseases. Six couples carrying chromosomal microdeletions associated with X-linked ichthyosis were recruited, and all couples entered the PGT process. Multiple displacement amplification (MDA) method was used to amplify the whole-genome DNA of trophectoderm cells. Then karyomapping based on single nucleotide polymorphism (SNP) was used for haplotype linkage analysis to detect alleles carrying microdeletions, and CNVs of embryos were identified to determine euploid identity. Amniotic fluid tests were performed in the second trimester to verify the PGT-M results. All couples were tested for chromosomal microdeletions, with deletion fragments ranging in size from 1.60 to 1.73 Mb, and one partner in each couple did not carry the microdeletion. Three couples successfully underwent PGT-M assisted conception and obtained healthy live births. This study shows that haplotype linkage analysis by karyomapping could effectively detect the carrier status of embryos with microdeletions at the single-cell level. This approach may be applied to the preimplantation diagnosis of various chromosomal microvariation diseases.

Enhancing the Genetic Diversity of FXI Deficiency: Structural Variants and New Cross Reactive Positive Variants

Introduction Factor XI (FXI) is a procoagulant element that circulates in plasma as a homodimer. It participates in both intrinsic and extrinsic coagulation pathways, as it is activated by both FXIIa and thrombin. Elevated levels of FXI are significantly associated with a high risk of thrombosis. FXI deficiency, considered a rare disorder in Caucasians, increases the risk of bleeding and protects against thrombosis. The identification of new naturally occurring genetic defects that cause FXI deficiency may help to identify new key domains and residues in the folding, secretion, dimerization and function of this molecule, as well as define its clinical impact. The aim of this study was to identify and characterize new genetic defects causing FXI deficiency. Methods We studied 91 unrelated Caucasian families with FXI deficiency (FXI:C<70%) identified mainly by prolonged aPTT. The entire F11 gene was sequenced by high-throughput sequencing (NGS). Structural variants (SVs), identified by MLPA, were characterized by nanopore long read sequencing using real-time enrichment methods on a MinION device, and validated by PCR and Sanger sequencing. Plasma FXI was characterized by functional methods using chromogenic and coagulation assays, and by immunological methods under both basal and silica-activated conditions. In addition, recombinant expression of wild-type (WT) and selected FXI variants was performed in HEK293. Results Thirty-one different F11 variants were identified. Three of them were novel SVs, being characterized at the nucleotide level by nanopore sequencing. They included two partial duplications, one involving exons 8-9 and the other exon 14; and one complete gene deletion spanning 7 MB including 142 genes. This was a de novo mutation originating in the paternal allele potentially during spermatogenesis. Interestingly, in all cases, repetitive elements were detected at the breakpoints. We also identified two small INDELs and 26 SNVs with different consequences (splicing defects, N=2; nonsense, N=3; and missense, N=21). We highlight 6 missense variants that, without affecting secretion (cross-reactive material, CRM+), produced FXI:C deficiency. Three of them were located in the catalytic domain; p.Pro538Leu, p.Glu565Lys and p.Cys599Tyr. Two variants disrupted the disulfide bridges p.Phe295Cys and p.Cys339Phe. The p.Phe295Cys was identified in a hemizygous state in a patient with severe FXI deficiency (FXI:C=4%). It produced two aberrant circulating FXI dimers through a potential shuffling of intramolecular disulfide bonds that were also observed in the recombinant protein. p.Cys339Phe affecting the residue involved in homodimerization was identified in 2 unrelated subjects with mild FXI deficiency. These individuals had FXI monomers in plasma that were activated by silica. Recombinant expression of this variant secreted only monomers, whereas WT expression in HEK293 secreted FXI dimers and monomers in the same proportion. The p.Arg268His variant located in the Apple 3 domain was identified in two subjects, one of them in homozygous state. Of note, we also detected 6 carriers of a variant affecting the same residue but causing CRM- deficiency (p.Arg268Cys). Most cases had not or only mild bleeding events which were not related to the mutation, type of deficiency, or FXI levels, although cases with severe deficiency presented more events. We point out a severe haemorrhage with fatal consequences during COVID-19 infection in a heterozyous carrier of the CRM+ p.Arg268His. Conclusions We confirm the genetic heterogeneity causing FXI deficiency in Caucasians. SVs affecting F11, probably caused by recombination of repetitive elements, are heterogeneous in type and extent and may not be detected by conventional short-read sequencing methods. Although most F11 variants cause type I defects, we have identified 6 type II or CRM+ deficiencies, and describe the first CRM+ variant affecting the Apple 3 domain. The data show that mutations in the catalytic domain or in aberrant disulfide bonds can allow secretion of inactive dimer variants. In addition, for the first time we describe the detection of monomeric variants in plasma. This, together with the similar secretion of WT FXI dimers and monomers in HEK293, suggests the relevance of Cys339 for the removal of WT monomers from the circulation.

A binary/digital approach to amino acid identification and its application to peptide sequencing and protein identification.

A binary/digital method is proposed in theory for the identification of single amino acids (AAs) in the bulk or with a few molecules from a single binary measurement. Combined with Edman degradation (or other cleaving method), it can be used to sequence a peptide or identify the parent protein from a partial sequence. The approach is centered on the superspecificity property of transfer RNAs (tRNAs). Markedly different from conventional and recent single molecule (SM) sequencing methods based on analog measurements, it changes the analytical question 'Which AA is it?' to the much simpler one 'Is there an AA in the detection space?'. Each of 20 terminal residues cleaved from 20 copies of a peptide enters a different cavity with a unique tRNA; tRNA charging (or binding with AA) occurs only in the cavity with the cognate AA. The bound AA or the AA separated from the tRNA is detected with a single binary measurement; its identity is known from the position of the single high bit in the resulting 20-bit output. Alternatively, a 20-stage pipeline can be used with sparse samples. Detection of the bound AA can be done optically by tagging the AAs with a fluorescent dye, or of the freed AA electrically with a nanopore. Necessary conditions for accurate AA identification are satisfied in principle; related computations and simulation results are presented. A modified version that can be used for de novo sequencing in parallel of large numbers of peptides immobilized on a glass slide with the tRNAs carrying a fluorescent tag is also proposed. Both methods can be used for protein identification from partial sequences containing 2 or 3 AA types by using only the corresponding tRNAs. Experiments may be performed to validate them, followed by translation into practice with existing technology; potential implementation issues are discussed. Binary/digital amino acid identification for peptide sequencing.

Performance Evaluation of the IR Biotyper® System for Clinical Microbiology: Application for Detection of Staphylococcus aureus Sequence Type 8 Strains.

Background: Methicillin-resistant S. aureus (MRSA) clonal lineages have been classified based on sequence type (ST) and pulsotype associated with human infection. Providing rapid and accurate epidemiological insight is important to address proper infection control in both community-acquired and nosocomial hospital settings. In this regard, this study was performed to evaluate the IR Biotyper® (IRBT®) for strain typing of S. aureus clinical isolates on three media. Methods: A total of 24 S. aureus clinical isolates comprising 15 MRSA isolates (six ST5, three ST72, three ST8, and three ST188 isolates) and nine methicillin-susceptible S. aureus (MSSA) isolates (three ST5, three ST72, and three ST8 isolates) were included for evaluating the IRBT®. Molecular characterization of all S. aureus isolates was performed by conventional PCR and sequencing methods. The IRBT® was evaluated according to manufacturer instructions and a modified sample procedure on commonly used BAP, MHA, and TSA media. Subsequently, the spectra obtained by IRBT® software were compared with dendrograms of PFGE analysis. Results: In this study, the modified sample procedure for reducing the amount of bacteria and bacterial concentration improved the acquisition quality pass rate of the IRBT®. Each spectrum of S. aureus ST5, ST72, and ST188 isolates on all three media could not be clustered by IRBT®. However, the dendrogram obtained from the spectra of S. aureus ST8 isolates on TSA medium were in concordance with that obtained by PFGE analysis. In addition, the visual distribution of S. aureus ST8 isolates on TSA medium in a 2D scatter plot appeared as separated point set from those of S. aureus ST5, ST72, and ST188 isolates. Conclusions: The IRBT® system is a rapid strain typing tool using the FTIR spectroscopic method. This system demonstrated the possibility of discriminating the strain types of S. aureus clinical isolates. Indeed, S. aureus ST8 isolates on TSA medium were successfully differentiated from other strain type isolates.

Genetic relationship of Staphylococcus aureus isolated from humans, animals, environment, and Dangke products in dairy farms of South Sulawesi Province, Indonesia.

Background and Aim:Staphylococcus aureus is a bacterium that causes several infectious diseases, including mastitis, endocarditis, and osteomyelitis, and poses a threat to human and animal health. This study aims to phenotypically and genetically identify S. aureus from the isolates collected from humans, animals, environment, and Dangke products in the dairy farms of South Sulawesi Province, Indonesia, as well as to establish a genetic relationship among the isolated S. aureus strains.Materials and Methods:The total number of samples was 142, comprising 30 humans (skin swab), 58 animals (raw milk), 14 dairy products (Dangke), and 40 environmental samples (water). S. aureus was phenotypically identified using the culture method, followed by Gram staining, catalase test, and coagulase test. Simultaneously, genotypic identification of S. aureus was performed using the conventional polymerase chain reaction and sequencing methods. Sequencing data were analyzed using the MEGA X software by comparing BLAST National Center for Biotechnology Information databases.Results:The phenotypic methods revealed that 56/142 (39.4%) animal, human, and Dangke samples grew on culture, and 56/56 (100%) were Gram stain positive, 56/56 (100%) catalase-positive, and 23/56 (41.1%) coagulase positive. The genotypic method revealed that 32/56 (57.1%) samples amplified the nuc gene. The phylogenetic analysis of 12 isolates revealed that they are all closely related and do not belong to distinct clades.Conclusion:It indicates that S. aureus isolates from animals (S30) are probably the same strain as human isolates (H2, H3, H4, and H5). The findings of this study can be used as information regarding the importance of preventing and controlling diseases caused by S. aureus using a health approach involving the human, animal, and environmental sectors. This study was limited to the sequencing analysis of the nuc gene.

High-throughput identification and quantification of single bacterial cells in the microbiota

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa–named herein cell-based operational taxonomy units (cOTUs)–based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

Challenges Related to the Use of Next-Generation Sequencing for the Optimization of Drug Therapy.

Over the last decade, next-generation sequencing (NGS) methods have become increasingly used in various areas of human genomics. In routine clinical care, their use is already implemented in oncology to profile the mutational landscape of a tumor, as well as in rare disease diagnostics. However, its utilization in pharmacogenomics is largely lacking behind. Recent population-scale genome data has revealed that human pharmacogenes carry a plethora of rare genetic variations that are not interrogated by conventional array-based profiling methods and it is estimated that these variants could explain around 30% of the genetically encoded functional pharmacogenetic variability.To interpret the impact of such variants on drug response a multitude of computational tools have been developed, but, while there have been major advancements, it remains to be shown whether their accuracy is sufficient to improve personalized pharmacogenetic recommendations in robust trials. In addition, conventional short-read sequencing methods face difficulties in the interrogation of complex pharmacogenes and high NGS test costs require stringent evaluations of cost-effectiveness to decide about reimbursement by national healthcare programs. Here, we illustrate current challenges and discuss future directions toward the clinical implementation of NGS to inform genotype-guided decision-making.

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.

Comparative analysis of alignment tools for application on Nanopore sequencing data

Abstract INTRODUCTION: Long-read sequencing techniques such as Oxford Nanopore sequencing, are representing a promising novel approach in molecular-biological methodology, enabling potential facilitation in mapping and de novo assembly. In comparison to conventional sequencing methods, novel alignment tools are mandated to compensate differing data structures (especially high error rate) to achieve acceptably accurate analysis results. METHODS: In this study, benchmarking for long read aligners BLASR, GraphMap, LAST, minimap2, NGMLR and the short-read aligner BWA MEM on three experimental datasets was conducted. Obtained alignment results were compared for various quality and performance criteria, such as match rate, mismatch rate, error rate, working memory usage and computational time. RESULTS: The comparison yielded differences in alignment quality and performance of tools under test. Tool LAST showed the largest differences among all tools. Minimap2 achieved constant quality with good performance. BLASR, GraphMap, BWA MEM and NGMLR showed slight differences only. CONCLUSION: Differences among the tools could be reasoned with dataset characteristics and algorithm approaches of individual tools. All tools except BLASR seem applicable for Nanopore sequencing data. Therefore, selection of the tool should be done under consideration of the experimental design and the further downstream analysis

Comparison of Conventional Molecular and Whole-Genome Sequencing Methods for Differentiating Salmonella enterica Serovar Schwarzengrund Isolates Obtained from Food and Animal Sources.

Over the last decade, Salmonella enterica serovar Schwarzengrund has become more prevalent in Asia, Europe, and the US with the simultaneous emergence of multidrug-resistant isolates. As these pathogens are responsible for many sporadic illnesses and chronic complications, as well as outbreaks over many countries, improved surveillance is urgently needed. For 20 years, pulsed-field gel electrophoresis (PFGE) has been the gold standard for determining bacterial relatedness by targeting genome-wide restriction enzyme polymorphisms. Despite its utility, recent studies have reported that PFGE results correlate poorly with that of closely related outbreak strains and clonally dominant endemic strains. Due to these concerns, alternative amplification-based molecular methods for bacterial strain typing have been developed, including clustered regular interspaced short palindromic repeats (CRISPR) and multilocus sequence typing (MLST). Furthermore, as the cost of sequencing continues to decrease, whole genome sequencing (WGS) is poised to replace other molecular strain typing methods. In this study, we assessed the discriminatory power of PFGE, CRISPR, MLST, and WGS methods to differentiate between 23 epidemiologically unrelated S. enterica serovar Schwarzengrund isolates collected over an 18-year period from distinct locations in Taiwan. The discriminatory index (DI) of each method for different isolates was calculated, resulting in values between 0 (not discriminatory) and 1 (highly discriminatory). Our results showed that WGS has the greatest resolution (DI = 0.982) compared to PFGE (DI = 0.938), CRISPR (DI = 0.906), and MLST (DI = 0.463) methods. In conclusion, the WGS typing approach was shown to be the most sensitive for S. enterica serovar Schwarzengrund fingerprinting.

Detection of SARS-CoV-2 spike protein D614G mutation by qPCR-HRM analysis

ObjectivesMonitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. MethodsSix SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. ResultsThe results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. ConclusionsThis study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

Determination of dermatophytes isolated from tinea capitis using conventional and ITS-based sequencing methods in Kano, Nigeria.

BackgroundTinea capitis is a dermatophyte infection of the scalp and hair that affects a large number of people worldwide. The disease commonly affects children and manifests with varying degrees of hair loss, scalp inflammation, and psychosocial impact. In Nigeria, the burden of tinea capitis is worrisome affecting over 15,000,000 school-age children. Molecular techniques complement the conventional mycological examinations in laboratory diagnosis of tinea capitis. In this study, we identified dermatophytes species causing tinea capitis in Kano, Nigeria, using ITS-based nucleotide sequencing technique in addition to conventional mycological examination. MethodsWe collected 112 samples from the scalp of children with clinically diagnosed tinea capitis at the dermatology clinic of Murtala Muhammad Specialist Hospital, Kano, between April and September 2019. The samples were processed and subjected to direct microscopy and mycological culture to isolate dermatophytes species that were identified morphologically and using ITS sequencing. ResultsOut of the 112 patients investigated, the majority (59.8%) were between the ages 6 and 9 years with a mean age of 7.3 ± 1.9 years. Males (79.5%) were predominantly affected. Black dot (46.4%) was the most common clinical type of tinea capitis followed by gray patch (39.3%) and kerion (1.8%). Favus was not observed. Microsporum audouinii (45.7%) was the predominant etiologic agent followed by Trichophyton soudanense (28.6%), T. violaceum (22.9%), and T. tonsurans (2.9%). ConclusionThe prominence of anthropophilic dermatophytes as the main causes of tinea capitis in our localities suggests that public health interventions to promote health education and good hygiene practices would minimize the transmission rate of tinea capitis among children in the study area.

Abstract PO-021: Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study

Abstract This study examines if single-cell DNA sequencing may be used to study the mutational effects of ionizing radiation. As the action of ionizing radiation is a stochastic process, each cell in an irradiated sample experiences its own unique radiation-induced DNA damage. As a result, conventional sequencing methods such as bulk cell sequencing cannot be used to identify individual mutations. In this work, Epstein-Barr virus (EBV) transformed B-lymphoblastoid cells were irradiated with 6 MV X-ray radiation using a medical linear accelerator. Four samples of cells from the same population (400,000 cells/ml) were exposed to sham irradiation (0 Gray [Gy]; control), 0.5 Gy, 1.5 Gy and 3.0 Gy respectively at a common dose rate of about 600 cGy/min. Irradiated samples were incubated for 24 hrs and subsequently underwent single-cell whole-genome DNA sequencing to characterize the radiation impact. Mutational profiles of approximately 500 cells, randomly selected from each sample, were individually analyzed and compared to identify the variation of several mutational parameters as a function of dose. We quantified the copy number variations (CNV) for each cell in our samples. Additionally, we segregated insertion CNVs and deletion CNVs and independently analyzed their dose dependences. We found that the total number of CNVs (insertion and deletion combined) increased with dose, and the number of deletion CNVs consistently increased most. We have repeated the experiment and a new round of single-cell DNA sequencing is underway. If confirmed, our results will demonstrate that the mutational effects of ionizing radiation may be examined directly using single-cell sequencing. Citation Format: Felix Mathew, Jonathan Yeo, Luc Galarneau, Norma Ybarra, Patricia Tonin, Yu Chang Wang, Ioannis Ragoussis, John Kildea. Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-021.