Sequencing Kit Research Articles

Single Laboratory Evaluation of the Q20+ Nanopore Sequencing Kit for Bacterial Outbreak Investigations

Leafy greens are a significant source of produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States, with agricultural water often implicated as a potential source. Current FDA outbreak detection protocols are time-consuming and rely on sequencing methods performed in costly equipment. This study evaluated the potential of Oxford Nanopore Technologies (ONT) with Q20+ chemistry as a cost-effective, rapid, and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could facilitate near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. High-quality, closed bacterial genomes were obtained from all combinations of extraction and sequencing kits. However, variations in assembly length and genome completeness were observed, indicating the need for further optimization. In silico analyses demonstrated that Q20+ nanopore sequencing chemistry accurately identified species, genotype, and virulence factors, with comparable results to Illumina sequencing. Phylogenomic clustering showed that ONT assemblies clustered with reference genomes, though some indels and SNP differences were observed, likely due to sequencing and analysis methodologies rather than inherent genetic variation. Additionally, the study evaluated the impact of a change in the sampling rates from 4 kHz (260 bases pair second) to 5 kHz (400 bases pair second), finding no significant difference in sequencing accuracy. This evaluation workflow offers a framework for evaluating novel technologies for use in surveillance and foodborne outbreak investigations. Overall, the evaluation demonstrated the potential of ONT Q20+ nanopore sequencing chemistry to assist in identifying the correct strain during outbreak investigations. However, further research, validation studies, and optimization efforts are needed to address the observed limitations and fully realize the technology’s potential for improving public health outcomes and enabling more efficient responses to foodborne disease threats.

Comparative analysis of whole exome sequencing kits for the canine genome.

As the public's interest in companion dogs grows, health issues in these animals are also emerging, necessitating the optimization of whole exome sequencing (WES) as a valuable method for disease prediction. While WES targeting the human genome is well established, WES targeting the canine genome is understudied, and there is a need to find effective analysis kits. We compared and analyzed the performance of three WES kits from Twist and Agilent using the canine genome as the target to perform genetic analysis of canine diseases effectively. The levels of total reads, the duplication rate, and variant calling in canine genomic DNA samples from seven healthy dogs (three beagles, one bichon fry, one maltese, one welsh corgi, and one mixed breed) without any interventions were examined through WES via Twist and Agilent kits. We found that while Twist had the lowest total read number, the number of reads in the SSXT series was significantly (P<0.05) greater. Twist showed low evenness and high standard deviation, but the SSXT series showed relatively high evenness. Compared with Twist, the SSXT series from a depth of 30× presented a significantly (P<0.05) greater target ratio. Among the four kits, the significantly lowest duplicate ratio was confirmed for SSXT (O/N) (30% lower than Twist). The most important performance of the kit, the number of variants detected, was 48,302 for Twist and 130,506 for SSXT (O/N). On the basis of the performance comparison results, SSXT (O/N) was found to have the best performance.

Amplicon-based long-read sequencing is useful for confirming zygosity of DSG2 variants associated with Japanese ARVC

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C&amp;gt;T (p.R292C) and c.1481A&amp;gt;C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B

Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of RNA from Escherichia coli isolates.

Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for reference-agnostic analysis of differential gene expression in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA- and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates per isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over eight flow cells using 16 barcodes to minimize batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance was quantified after in silico depletion of low-abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted that correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC content.

Revolutionising High Resolution HLA Genotyping for Transplant Assessment: Validation, Implementation and Challenges of Oxford Nanopore Technologies' Q20+ Sequencing.

The advent of third-generation sequencing (TGS) represents a significant shift in the field of genetic sequencing, enabling single-molecule sequencing to overcome limitations of short-read NGS platforms. Several studies have assessed the utilisation of TGS in HLA genotyping, though many of these studies have described the high error rate as an obstacle to achieving a robust and highly accurate HLA typing assay. In 2021, Oxford Nanopore Technologies (ONT) released the high-accuracy sequencing Kit 14 and the MinION flow cell model R10.4.1, which were reported to achieve sequencing accuracies up to 99%. The aim of this study was to validate this novel high-accuracy sequencing kit for HLA genotyping coupled with a full-gene HLA PCR assay. Comparison with historical data obtained using legacy flow cell models such as R9.4, R10.3 and R10.4 was also done to assess progressive improvement in sequencing performance with each sequential release. The workflow was validated based on data throughput, sequence quality and accuracy, and HLA genotyping resolution. An initial validation was performed using an internal reference panel of 42 samples representing common HLA allele groups, followed by an analysis of data obtained from 111 sequencing batch runs since the implementation, to assess assay performance and define quality control metrics to assess inter-run variability and monitor quality. Furthermore, challenges arising from MinION flow cell stability and use, and assessment of barcode contamination are discussed. The findings of this study highlight advantages of ONT sequencing kit 14/R10.4.1 for HLA genotyping and the implementation considerations for the routine diagnostic HLA laboratory.

A Comparison of Sanger Sequencing and Amplicon-Based Next Generation Sequencing Approaches for the Detection of HIV-1 Drug Resistance Mutations.

Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. The samples had a median 4.8 log [IQR 4.4-5.2] HIV-1 RNA copies/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2/28) compared with AD4SEQ (13/28) (p < 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13/28 and 9/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage.

Discordance of Penta D x.4 microvariant alleles results between three capillary electrophoresis and one massively parallel sequencing short tandem repeat kits

Forensic Biology is contingent upon matching DNA profiles between a crime sample and a reference sample. There are several capillary electrophoresis kits available to generate a short tandem repeat (STR) profile from DNA samples, while newer methods using massively parallel sequencing are slowly being implemented in forensic laboratories worldwide. During evaluation of a newer capillary electrophoresis kit, Applied Biosystems™ VeriFiler™ Plus, a discordance was observed in the Penta D locus. The previous kit, Promega PowerPlex 21® System produced a 13.4,14 genotype, whilst VeriFiler™ Plus produced a 14,14 genotype. An expanded investigation into Penta D microvariant alleles revealed that multiple discordances were observed for DNA profiles containing larger x.4 variants. There was full concordance between PowerPlex® 21 and QIAGEN Investigator® 26plex, however discordances were observed between VeriFiler™ Plus and the other three kits tested, including the massively parallel sequencing kit, Verogen ForenSeq® MainstAY. Notably, four of these discordances resulted in null alleles with the VeriFiler™ Plus kit. A review of the Penta D DNA sequences in MainstAY revealed fully concordant microvariant alleles involved deletions within the repeat region, whilst variability in the discordances observed were dependent on the location of the variation outside the repeat region and the analysis method used. Variations observed within the 5’ flanking region produced the same allele designation across all capillary electrophoresis kits. However, deletions within the 3’ region either produced a null allele for VeriFiler™ Plus where the deletion is thought to overlap the primer binding site, or microvariant alleles for the PowerPlex® 21 and Investigator 26plex kits, which produced longer Penta D amplicons. The discovery of these variations in the Penta D flanking sequences is informative as it increases the awareness of Penta D discordances between different kit chemistries in nominated reference DNA profile comparisons and DNA database searching and matching alike, and provides support for this phenomenon when providing evidence as to the admissibility of such results in trial proceedings.

Unraveling changes in the duck microbiome and inflammatory processes due to allithiamine-enriched feed

The gastrointestinal tract of poultry harbors a diverse and intricate microbiome that plays a crucial role in nutrient digestion and absorption, immune system development, and enhances resistance against pathogens. Maintaining a healthy state and proper production is fundamentally determined by the symbiosis between the host and microbes. Due to genetic and technological improvements, intensive growth rate can be associated with many pathological conditions, such as increased susceptibility to infections. Intestinal inflammation in poultry industries detrimentally affects productivity by hindering nutrient absorption and the efficient allocation of nutrients for growth. The host releases different biomarkers in response to inflammation. Hence, there is an utmost interest of reliable, precise, sensitive and robust biomarkers to evaluate both the gastrointestinal health status and inflammation in poultry. The aim of this study was to determine how the developed feed prototype (allithiamine) affects the community diversity in raised duck, and the relationship between gut microbiome composition and inflammatory factor as calprotectin, using targeted 16S rRNA gene amplicon sequencing and Chicken Calprotectin ELISA Kit.

Sequencing accuracy and systematic errors of nanopore direct RNA sequencing

BackgroundDirect RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied.ResultsWe present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits.ConclusionsAs the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.

Establishing national reference materials for genetic testing of cytochrome P450.

Reference materials for in-vitro diagnostic reagents play a critical role in determining the quality of reagents and ensuring the accuracy of clinical test results. This study aimed to establish a national reference material (NRM) for detecting cytochrome P450 (CYP) genes related to drug metabolism by screening databases on the Chinese population to identify CYP gene polymorphism characteristics. To prepare the NRM, we used DNA extracted from healthy human immortalized B lymphoblastoid cell lines as the raw material. Samples of these cell lines were obtained from the Chinese Population PGx Gene Polymorphism Biobank. Further, we used Sanger sequencing, next-generation sequencing, and commercial assay kits to validate the polymorphic genotypes. Among the CYP superfamily genes, we confirmed 24 riboswitch loci related to drug metabolism, with evidence levels of 1A, 2A, 3, and 4. We confirmed the polymorphic loci and validated their genotypes using various sequencing techniques. Our results were consistent with the polymorphism information of samples obtained from the biobank, thus demonstrating high precision and stability of the established NRM. An NRM (360 056-202 201) for CYP genetic testing covering 24 loci related to drug metabolism was established and approved to assess in-vitro diagnostic reagents containing CYP family gene polymorphisms and perform clinical inter-room quality evaluations.

Sequencing complex plants on a budget: The development of Kalanchoë blossfeldiana as a C3, CAM comparative tool

Societal Impact StatementResearch efforts in plant biology have often been focused on sequenced and well‐studied ‘model’ organisms. Despite the advent of relatively inexpensive genome sequencing, most plant taxonomic groups are underrepresented, with few species that ‘represent’ the diversity of whole genera. This study describes an economical guide to sequencing a non‐model organism, which may be useful in reducing the cost of sequencing more species within genera and across plant life. This method was used to develop Kalanchoë blossfeldiana as a resource for comparing C3 and the water‐conserving mode of photosynthesis known as Crassulacean acid metabolism (CAM) within the same plant.Summary Despite the increasing number of well‐studied plant species with well‐annotated genomes across plant life, there are few densely sampled genera with more than a couple of genome sequences representing the diversity of whole genera. Here, we develop an economic approach to full‐genome sequencing that could be used to sequence many species within a genus. We made use of the Nanopore rapid sequencing kit to assist in plant genome assembly, dramatically reducing the cost. Here we applied this method to cost‐effectively develop genomic resources for Kalanchoë blossfeldiana, a commercially important ornamental, in which Crassulacean Acid Metabolism (CAM), a water‐conserving mode of photosynthesis can be induced. We present a physiological and biochemical characterisation of Kalanchoe blossfeldiana with its nuclear and chloroplastic genome and a comparative C3, CAM dusk transcriptome. We apply this approach to a complex tetraploid genome, making use of a relative species for chromosomal scaffolding to reduce assembly ploidy, we provide a resource for future gene expression studies. We highlight its limitations, e.g. the need for deeper sequencing to accurately resolve genome structure and haplotypes without using a relative species for scaffolding. The study demonstrates the merits of K. blossfeldiana as a comparative system for studying C3 and CAM within a plant and has identified substantial changes in the dusk transcriptome between young C3 and mature CAM K. blossfeldiana leaves in response to age‐induced CAM, and shows that in the absence of abiotic stress, CAM induction still involves the engagement of drought and abscisic acid (ABA) response pathways.

Developmental validation of the ForenSeq® Kintelligence kit, MiSeq FGx® sequencing system and ForenSeq Universal Analysis Software

Forensic Investigative Genetic Genealogy, a recent sub discipline of forensic genomics, leverages the high throughput and sensitivity of detection of next generation sequencing and established genetic and genealogical approaches to support the identification of human remains from missing persons investigations and investigative lead generation in violent crimes. To facilitate forensic DNA evidence analysis, the ForenSeq® Kintelligence multiplex, consisting of 10,230 SNPs, was developed. Design of the ForenSeq Kintelligence Kit, the MiSeq FGx® Sequencing System and the ForenSeq Universal Analysis Software is described. Developmental validation in accordance with SWGDAM guidelines and forensic quality assurance standards, using single source samples, is reported for the end-to-end workflow from library preparation to data interpretation. Performance metrics support the conclusion that more genetic information can be obtained from challenging samples compared to other commercially available forensic targeted DNA assays developed for capillary electrophoresis (CE) or other current next generation sequencing (NGS) kits due to the higher number of markers, the overall shorter amplicon sizes (97.8% <150 bp), and kit design. Data indicate that the multiplex is robust and fit for purpose for a wide range of quantity and quality samples. The ForenSeq Kintelligence Kit and the Universal Analysis Software allow transfer of the genetic component of forensic investigative genetic genealogy to the operational forensic laboratory.

Interplay between green-synthesized nanoparticles and plant performance is mediated by the microbial community in the rhizocompartments

Numerous green-synthesized nanoparticles are being evaluated for their potential application in soil, yet understanding their influence on plant performance and their rhizocompartments associated microbes is a gap that needs to be addressed. In this study, we investigated how applying iron oxide nanoparticles (control 0, low 25 and high 50 mg/kg) impacted the rhizocompartments associated microbial communities and plant secondary metabolites grown in natural soil microcosm. The transcriptional analysis showed that the majority of artemisinin and flavonoid biosynthetic pathway genes were significantly upregulated in plants with the application of low concentration of green-synthesized iron oxide nanoparticles (FeO.NPs) when compared with control plants, suggesting a fine collaboration between these metabolites’ pathways, as indicated by a remarkable increase in artemisinin content (2-fold), ferulic acid (7.6-fold), luteolin (4.3-fold) and syringic acid (6.2-fold). The response of rhizocompartments microbial communities and associated soil enzymes was investigated using high-throughput sequencing and profiler test kits, respectively. The soil microbial community structure was altered upon FeO.NPs exposure in both low and high treated soils as indicated by multivariate analysis. For instance, bacterial phyla Bacteroidetes and Gemmatimonadetes and fungal phyla Ascomycota demonstrated a significant rise in relative abundance in both the rhizosphere and rhizoplane in low and high treatments relative to the control. Moreover, genera level analysis revealed that Cupriavidus, Vibrionimonas, and Burkholderia displayed higher relative abundance (p < 0.05) in low FeO.NPs concentration while Giaella and Nonomuraea didn't show any noticeable response to FeO.NPs. In terms of fungal genera, Oidiodendron and Cryptococcus showed high relative abundance under low treatment. The altered microbiome profile is possibly associated with soil enzymes, for instance, urease and catalase activities were increased by 1.76 and 1.71-fold, respectively in low FeO.NPs. Thus, this study demonstrates the effects of FeO.NPs gradients on plant secondary metabolites and soil enzyme activities, while contributing new insights into their specific impacts on the microbial community composition in the rhizocompartments of plant root system.

Genetic Diversity of Mangrove and Nypa Palm Species in the Niger Delta, Nigeria

Mangroves and nypa palm are two dominant coastal vegetation in the Niger delta that are sources of numerous ecosystem services. We thus hypothesized that both species are monophyletic, which means they are identical by descent. We began the study by randomly collecting fresh leave samples of nypa palm in a mixed forest comprising nypa palm and mangrove at a coastal community called Eagle Island. We put the leaves samples in zip lock bags containing silica gel crystals and transported them to the laboratory, where the DNA of the leaves was extracted. A quick-DNA plant/seed miniprep kit (Zymo-research Lab, California, USA). A total of six primer pairs were used for the amplification, namely: three microsatellite markers and three universal primers. Fragments of the samples were sequenced using the Ninagen, Brilliant DyeTM Terminator Cycle Sequencing kit V3.1 BRD3-100/1000. All three samples of the nypa palm were amplified, and the BLAST prediction tool was used to produce the new species. Our results show that out of the three samples sequenced (NI, N2, and N3), the N1 sample was identified as Nypa fruticans with 100% identity (Accession Number is KT312925.1), while the others were the new strains identified in the study namely N2 identified as Astrocaryum aculeatum with 98.95% identity (Accession Number is NC 044482.1) and N3 identified as Astrocaryum aculeatum with 99.21% (Accession Number is 044482.1).

Molecular Identification of Dominant Microbes in Kola nut (Cola nitida)

Kola nut is an important dehiscent fruit but its supply fails to meet the demand due to limitations such as inadequate post-harvest practices leading to spoilage during transportation and storage. Identification of microbial contaminants is a necessary step in reducing the loss. Hence, this work aimed at characterizing the genome of dominant fungus and bacteria associated with kola nut (Cola nitida). Samples of kola nuts were collected from Oje and Oja-oba markets in Ibadan, Nigeria. Isolates were extracted from the lesion region of the samples using the standard method. Macro-morphological and micro-morphological observations of the pure culture of the isolates were conducted. After genomic DNA extraction of fungal and bacterial isolates, Polymerase Chain Reaction (PCR) was carried out using Internal Transcribed Sequence (ITS 1 and ITS 4) and 27F primers for fungal and bacterial genome respectively. Sanger sequencing of the purified DNA fragments was conducted utilizing the Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000. Molecular Evolutionary Genetics Analysis (MEGA version 6) was used for phylogenetic analysis. The result revealed that one fungus and two bacteria were associated with kola nuts. The fungal isolate showed 100% identity with Aspergillus wentii from the National Center for Biotechnology Information data base. The fungus showed a good relationship with A. dimorphicus and A. europaeus. The two bacterial isolates were also identified through the phylogenic analysis as Bacillus cereus and B. subtilis. B. cereus is closely related to B. thuringiensis. This research provides background information on the Kola nut microbial contaminant. Keywords: Bacteria, Contaminant, Fungus, Identification, Primers, Sequencing

Using nanopore sequencing to identify bacterial infection in joint replacements: a preliminary study.

This project investigates if third-generation genomic sequencing can be used to identify the species of bacteria causing prosthetic joint infections (PJIs) at the time of revision surgery. Samples of prosthetic fluid were taken during revision surgery from patients with known PJIs. Samples from revision surgeries from non-infected patients acted as negative controls. Genomic sequencing was performed using the MinION device and the rapid sequencing kit from Oxford Nanopore Technologies. Bioinformatic analysis pipelines to identify bacteria included Basic Local Alignment Search Tool, Kraken2 and MinION Detection Software, and the results were compared with standard of care microbiological cultures. Furthermore, there was an attempt to predict antibiotic resistance using computational tools including ResFinder, AMRFinderPlus and Comprehensive Antibiotic Resistance Database. Bacteria identified using microbiological cultures were successfully identified using bioinformatic analysis pipelines. Nanopore sequencing and genomic classification could be completed in the time it takes to perform joint revision surgery (2-3h). Genomic sequencing in this study was not able to predict antibiotic resistance in this time frame, this is thought to be due to a short-read length and low read depth. It can be concluded that genomic sequencing can be useful to identify bacterial species in infected joint replacements. However, further work is required to investigate if it can be used to predict antibiotic resistance within clinically relevant timeframes.

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology

A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Abstract A028: Quantifying resistance to targeted anticancer therapies at scale with duplex sequencing

Abstract Drug resistance to targeted anticancer therapeutics is one of the most researched problems in cancer genetics. A fundamental limiting factor facing most drug resistance studies is that resistance is studied retrospectively, i.e., mutants are characterized for their drug resistance only once they are seen in the clinic. Deep mutational scanning (DMS) is a field of functional genomics that can enable a-priori identification of resistant mutants- long before they are seen in the clinic. Quantifying resistance prospectively can guide clinical treatment decision-making and drug discovery efforts. In order to guide clinical practice, a DMS screen would need to have the sensitivity to measure drug resistance well and be able to relate screen measurements to clinical pharmacology. However, current DMS screens are not adopted for drug resistance and face unique technical challenges that limit the overall interpretability of screen data. Here we demonstrate the first scalable duplex sequencing DMS screen and use it to study Imatinib resistance in BCR-ABL. We first studied the pooled growth of 20 known Imatinib resistant mutants at low allele frequencies. We observed significant depletion artifacts and dose-response variations when measuring mutant outgrowth using allele frequency alone. To correct for these artifacts, we developed an inference-based approach that identifies and addresses depletion and dose variation artifacts that are commonly seen in drug resistance screens. The described measurement methodology enables precise and accurate tracking of low allele-frequency mutants. We also describe the first error-corrected duplex sequencing workflow that can achieve sequencing depths of 3 × 105, which is 20x higher than commercially available duplex sequencing kits. Sequencing at these depths enabled us to detect and measure 96.7% of all possible amino acid substitutions in the ABL kinase (n=4,788 mutants). We used our measurements to characterize all treatment-emergent variants of uncertain drug resistance in CML patients. We find that 22% of the rare variants seen in Imatinib refractory CML patients drive imatinib resistance, while the rest are likely bystanders of another resistance mechanism. Furthermore, because we can relate our measurements to clinical pharmacology, we identify variants that are good candidates for dose escalation from 400mg to 800mg daily dose of imatinib. Here, we demonstrate the first scalable duplex sequencing DMS screen that achieves high levels of sensitivity and specificity in measuring mutant drug resistance. We demonstrate the utility of this workflow by characterizing all variants of uncertain drug resistance in the ABL kinase. Addressing technical key barriers that face DMS screens of drug resistance enables us to make recommendations for treatment decisions on variants of unknown drug resistance. This approach can be used by pharmaceutical companies and geneticists to prospectively identify drug resistant variants to investigational drugs in candidate druggable biomarkers. Citation Format: Haider Inam, Josh Reynolds, Marta Tomaszkiewicz, Scott Leighow, Yiyun Rao, Ivan Sokirniy, Justin Pritchard. Quantifying resistance to targeted anticancer therapies at scale with duplex sequencing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A028.

First whole-genome sequence and assembly of the Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps), a critically endangered species, using Oxford Nanopore Technologies.

The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as critically endangered [International union for conservation of nature (IUCN)]. It faces multiple threats, the most significant one being habitat loss due to deforestation in western Ecuador. Genomic tools are keys for the management of endangered species, but this requires a reference genome, which until now was unavailable for A. f. fusciceps. The present study reports the first whole-genome sequence and assembly of A. f. fusciceps generated using Oxford Nanopore long reads. DNA was extracted from a subadult male, and libraries were prepared for sequencing following the Ligation Sequencing Kit SQK-LSK112 workflow. Sequencing was performed using a MinION Mk1C sequencer. The sequencing reads were processed to generate a genome assembly. Two different assemblers were used to obtain draft genomes using raw reads, of which the Flye assembly was found to be superior. The final assembly has a total length of 2.63 Gb and contains 3,861 contigs, with an N50 of 7,560,531 bp. The assembly was analyzed for annotation completeness based on primate ortholog prediction using a high-resolution database, and was found to be 84.3% complete, with a low number of duplicated genes indicating a precise assembly. The annotation of the assembly predicted 31,417 protein-coding genes, comparable with other mammal assemblies. A reference genome for this critically endangered species will allow researchers to gain insight into the genetics of its populations and thus aid conservation and management efforts of this vulnerable species.