Ion Torrent Sequencing Research Articles

Effect of temperature variations in anaerobic fluidized membrane bioreactor: membrane fouling and microbial community dynamics assessment

A single-stage anaerobic fluidized membrane bioreactor (AnFMBR) was applied to investigate the effects of temperature changes on membrane fouling while treating real municipal wastewater. The AnFMBR was operated at four temperature phases: 25 °C for 42 days, 20 °C for 20 days, 15 °C for 15 days, and at 10 °C for 15 days. The systems achieved a total chemical oxygen demand (TCOD) removal efficiency of above 90% at all phases. As temperature decreased, accumulation of solids and possible incomplete hydrolysis led to an increase in TCOD and volatile fatty acids (VFAs) in the reactor. However, as temperature reduced to 10 °C, VFAs in the reactor reduced probably an indication of reactors adaptation. Total membrane filtration resistance gradually increased to 1.1 × 1011 m−1 from 2.1 × 1009 m−1 with a temperature decrease from 25 °C to 10 °C. This corresponded to a significant decrease in membrane permeability from 1.68 to 0.05 LMH/kpa. The protein fraction of the extracellular polymeric substances (EPS) was dominant in all phases, which was ascribed for significant membrane fouling causing permeability deterioration. Microbial richness and diversity analysis using next generation Ion torrent sequencing methods revealed that Proteobacteria phylum was most dominant at 25 °C, whereas Bacteroidetes, which are responsible for releasing proteinaceous EPS, were most dominant at low temperatures (15 °C and 10 °C), contributing to severe fouling. In conclusion, decrease in temperature did not affect the treatment efficiency but resulted in gradual increase in membrane fouling.

27. Review and comparison of the Oncomine Myeloid Assay GX v2 on Genexus System

Rapid molecular results for FLT3, IDH1/2 and NPM1 are necessary for treatment decisions of newly diagnosed acute leukemia patients. However, as treatment protocols and clinical trials request more gene variants with a faster turnaround time, single gene STAT testing using polymerase chain reaction-based testing becomes more cumbersome for the molecular diagnostic laboratory. Therefore, a rapid next generation sequencing panel for acute leukemias is necessary for laboratory operations and, more importantly, clinical utility. Cedars-Sinai molecular pathology laboratory validated the automated Oncomine? Myeloid Assay GX v2 panel using the Ion Torrent? Genexus? Integrated Sequencer (v.6.6) for a 24-hour turnaround time from DNA and RNA purification to variant reporting. We compared the results from Oncomine? Myeloid Assay GX v2 panel to Archer® VariantPlex® Myeloid panel on Illumina-based platforms. The differences between the two systems include technician intervention, turnaround time, variant accuracy, and the usability of the software package. While Archer® VariantPlex® Myeloid panel demonstrates unrivaled accuracy and reliability, the ThermoFisher Myeloid Assay GX v2 using the Genexus (IonTorrent sequencing) offers quick results and fusion detection. Therefore, we have chosen to implement both systems as standard clinical diagnostics for new acute leukemia patients.

Investigating the Origin of Mycobacterium chimaera Contamination in Heater-Cooler Units: Integrated Analysis with Fourier Transform Infrared Spectroscopy and Whole-Genome Sequencing.

Mycobacterium chimaera is ubiquitously spread in the environment, including factory and hospital water systems. Invasive cases of M. chimaera infection have been associated with aerosols produced by the use of heater-cooler units (HCU) during cardiac surgery. The aim of this study was to evaluate for the first time the performance of IR-Biotyper system on a large number of M. chimaera isolates collected from longitudinal environmental HCUs samples and water sources from hospitals located in three Italian provinces. In addition, IR-Biotyper results were compared with whole-genome sequencing (WGS) analysis, the reference method for molecular epidemiology, to investigate the origin of M. chimaera contamination of HCUs. From November 2018 to May 2021, 417 water samples from 52 HCUs (Stockert 3T, n = 41 and HCU40, n = 11) and 23 hospital taps (used to fill the HCU tanks) were concentrated, decontaminated, and cultured for M. chimaera. Positive cultures (n = 53) were purified by agar plate subcultures and analyzed by IR-Biotyper platform and Ion Torrent sequencing system. IR-Biotyper spectra results were analyzed using a statistical approach of dimensionality reduction by linear discriminant analysis (LDA), generating three separate clusters of M. chimaera, ascribable to each hospital. Furthermore, the only M. chimaera-positive sample from tap water clustered with the isolates from the HCUs of the same hospital, confirming that the plumbing system could represent the source of HCU contamination and, potentially, of patient infection. According to the genome-based phylogenies and following the classification proposed by van Ingen and collaborators in 2017, three distinct M. chimaera groups appear to have contaminated the HCU water systems: subgroups 1.1, 2.1, and branch 2. Most of the strains isolated from HCUs at the same hospital share a highly similar genetic profile. The nonrandom distribution obtained with WGS and IR-Biotyper leads to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, in addition with the current hypothesis that contamination occurs at the HCU production site. This opens the possibility that other medical equipment, such as endoscope reprocessing device or hemodialysis systems, could be contaminated by M. chimaera. IMPORTANCE Our manuscript focuses on interventions to reduce waterborne disease transmission, improve sanitation, and control infection. Sanitary water can be contaminated by nontuberculous Mycobacteria, including M. chimaera, a causative agent of invasive infections in immunocompromised patients. We found highly similar genetic and phenotypic profiles of M. chimaera isolated from heater-cooler units (HCU) used during surgery to thermo-regulate patients' body temperature, and from the same hospital tap water. These results lead to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, adding to the current hypothesis that contamination occurs at the HCU production site. In addition, this opens the possibility that other medical equipment using sanitized water, such as endoscope reprocessing devices or hemodialysis systems, could be contaminated by nontuberculous Mycobacteria, suggesting the need for environmental surveillance and associated control measures.

Wild Bird Surveillance in the Gauteng Province of South Africa during the High-Risk Period for Highly Pathogenic Avian Influenza Virus Introduction.

Migratory birds carried clade 2.3.4.4B H5Nx highly pathogenic avian influenza (HPAI) viruses to South Africa in 2017, 2018 and 2021, where the Gauteng Province is a high-risk zone for virus introduction. Here, we combined environmental faecal sampling with sensitive rRT-PCR methods and direct Ion Torrent sequencing to survey wild populations between February and May 2022. An overall IAV incidence of 42.92% (100/231) in water bird faecal swab pools or swabs from moribund or dead European White Storks (Ciconia ciconia) was detected. In total, 7% of the IAV-positive pools tested H5-positive, with clade 2.3.4.4B H5N1 HPAI confirmed in the storks; 10% of the IAV-positive samples were identified as H9N2, and five complete H9N2 genomes were phylogenetically closely related to a local 2021 wild duck H9N2 virus, recent Eurasian LPAI viruses or those detected in commercial ostriches in the Western and Eastern Cape Provinces since 2018. H3N1, H4N2, H5N2 and H8Nx subtypes were also identified. Targeted surveillance of wild birds using environmental faecal sampling can thus be effectively applied under sub-Saharan African conditions, but region-specific studies should first be used to identify peak prevalence times which, in southern Africa, is linked to the peak rainfall period, when ducks are reproductively active.

Genomic diversity and molecular epidemiology of a multidrug-resistant Pseudomonas aeruginosa DMC30b isolated from a hospitalized burn patient in Bangladesh

Objectives: Pseudomonas aeruginosa is a key opportunistic pathogen causing a wide range of community- and hospital-acquired infections in immunocompromised or catheterized patients. Here, we report the complete genome sequence of a multidrug-resistant (MDR) P. aeruginosa DMC30b to elucidate the genetic diversity, molecular epidemiology, and underlying mechanisms for antimicrobial resistance and virulence. Methods: P. aeruginosa DMC30b was isolated from septic wound swab of a severe burn patient. Whole-genome sequencing was performed under Ion Torrent platform. The genome was assembled using the SPAdes v. 3.12.01 in an integrated Genome Analysis Platform for Ion Torrent sequence data. The genome was annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline. In-silico predictions of antimicrobial resistance genes, virulence factor genes, and metabolic functional potentials were performed using different curated bioinformatics tools. Results: P. aeruginosa DMC30b was found as a MDR strain and belonged to sequence type 244 (ST244). The complete genome size is 6 994 756 bp with a coverage of 76.76x, guanine-cytosine content of 65.7% and a Benchmarking Universal Single-Copy Orthologs score of 100. The genome of P. aeruginosa DMC30b harboured two predicted plasmid replicons (e,g. IncP-6; 78 007 bp and ColRNAI; 9359 bp), 35 resistomes (antimicrobial resistance genes) conferring resistance to 18 different antibiotics (including four beta-lactam classes), and 214 virulence factor genes. It was identified as the 167th ST244 strain among ∼ 5800 whole-genome sequences of P. aeruginosa available in the NCBI database. Conclusion: The MDR P. aeruginosa DMC30b was identified as the 167th ST244 complete genome to be submitted to the NCBI, and the first ST244 isolate sequenced from Bangladesh. The complete genome data with high genetic diversity and underlying mechanisms for antimicrobial resistance and virulence of P. aeruginosa DMC30b will aid in understanding the evolution and phylogeny of such high-risk clones and provide a solid basis for further research on MDR or extensively drug resistant strains.

Abstract 1551: miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel

Abstract Although a majority of women with high-grade serous ovarian cancer (HGSC) achieve a complete response to first-line platinum- and paclitaxel-based chemotherapy, around thirty percent of patients are identified as incomplete responders. Their diseases typically either does not respond or progresses during treatment (refractory) or recurs within 6 months of completing treatment (resistant), suggesting the inherit or intrinsic characteristics of the tumor. Currently, there are no biomarkers that can predict intrinsic resistance, which can avoid unnecessary treatment and toxicity. MicroRNAs (miRNAs) provide a novel master layer of regulation of gene expression. However, the molecular mechanisms by which miRNAs confer a chemoresistant phenotype in ovarian cancer have not been elucidated. To identify miRNAs that are associated with intrinsic chemoresistance in HGSC, Ion Torrent next-generation sequencing analysis of miRNAs was performed on microdissected chemosensitive and chemorefractory primary HGSC cases. Amongst all the miRNAs that were significantly down-regulated in chemorefractory cases, lower miR-625-3p expression was found to be associated with poorer overall and progression-free survivals. Further functional studies showed that overexpression of miR-625-3p significantly decreased cisplatin and paclitaxel resistance in ovarian cancer cells in vitro and in vivo. Online target prediction algorithms together with transcriptome profiling of HGSC cells transfected with miR-625-3p mimics or control mimics identified SSX2IP as a direct target of miR-625-3p, suggesting that SSX2IP may play a role in conferring cisplatin/paclitaxel resistance in HGSC by mediating the effect of miR-625-3p. SSX2IP is a microtubule anchoring and centriolar satellite protein, which plays a critical role in controlling microtubule length and orientation, and centrosome maturation. We demonstrated that HGSC cells transfected with SSX2IP had marked increases in microtubule masses, intracellular multivesicular body trafficking and exosomal cisplatin level; and a decrease in intracellular cisplatin level. In conclusion, down-regulation of miR-625-3p confers cisplatin/paclitaxel resistance in ovarian cancer cells via its novel direct target SSX2IP. SSX2IP alters the microtubule dynamics and subsequently mediates inter-organelle crosstalks between microtubules and multivesicular bodies, which facilitate exosomal export of cisplatin and paclitaxel of HGSC cells. This study is crucial for developing new predictive biomarkers for intrinsic chemoresistance, and new treatment strategies for HGSC based on upregulating miR-625-3p or downregulating SSX2IP expression in ovarian cancer cells, which will enhance cisplatin and paclitaxel sensitivity, and improve patient survival rates. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Kay-Pong Yip, Sammy Ferri-Borgogno, Samuel Mok. miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1551.

Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked.

Analysis of the methods and quality assurance of metagenomic next-generation sequencing to detect the microbial cfDNA from blood samples in China

Objective: To investigate the methods and quality assurance of metagenomic next-generation sequencing (mNGS) to detect the microbial cfDNA (mcfDNA) from blood samples in different laboratories across China. Methods: In October 2020, questionnaires about detecting mcfDNA in blood samples with mNGS were distributed to 80 laboratories across the country. The questionnaire included four parts: pre-analysis, during analysis, post-analysis, and carrying out of performance validation for mNGS. (1) Pre-analysis: the requirements for samples quality, such as collection, storage, the transportation conditions of samples; (2) During analysis: the extraction workflows of mcfDNA, the quality requirements of the library, the application of the sequencing platforms and the bioinformatics analysis pipelines; (3) Post-analysis: the standard of interpretation results for mNGS; (4) Carrying out of performance validation: the minimum detection limit for various pathogens. All laboratories are required to fill in the questionnaire according to the actual situation. The feedback data were summarized and analyzed. Results: The 80 laboratories included 20 medical centers and 60 independent medical laboratories. There were 80.0% (64/80) of laboratories indicated that both plasma and serum samples were used to detect mcfDNA in blood, and the rest of the laboratories (16/80, 20.0%) only used plasma samples. The sequencing platforms used by mNGS laboratories involved in the survey included illumina (49), Beijing Genomics Institute (16), Ion Torrent (13) and Nanopore sequencing (2). There were 87.5% (70/80) of laboratories used the integrated analysis tools built by the third-party laboratories, and other laboratories (12.5%, 10/80) independently built the analysis platform by open-source software. The interpretation criteria of mNGS results varied between laboratories, among which the normalized number of pathogen-specific sequences, relative abundance, genome coverage rate, and the detection of the microorganism in the negative control were the main factors considered by laboratories. Most laboratories (76.3%, 61/80) had carried out the performance validation for the mcfDNA mNGS workflows. The limit of detection of the laboratories-developed mNGS workflows for Gram-positive bacteria, Gram-negative bacteria, fungi, parasites, and other pathogens were mainly distributed at 10-100 copies/ml, DNA virus was mainly distributed at 500-1 000 copies/ml. Conclusions: The mNGS workflows of various laboratories are very different. In order to ensure timely and accurate testing results, every laboratory needs to actively optimize the mNGS testing procedures, improve quality assurance measures, and carry out performance validation before mNGS is widely used in clinical settings.

Novel hybridization- and tag-based error-corrected method for sensitive ctDNA mutation detection using ion semiconductor sequencing

Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.

Detection of genetic mutations in patients with breast cancer from Saudi Arabia using Ion AmpliSeq™ Cancer Hotspot Panel v.2.0.

Next-Generation Sequencing allows for quick and precise sequencing of multiple genes concurrently. Recently, this technology has been employed for the identification of novel gene mutations responsible for disease manifestation among breast cancer (BC) patients, the most common type of cancer amongst Arabian women, and the major cause of disease-associated death in women worldwide. Genomic DNA was extracted from the peripheral blood of 32 Saudi Arabian BC patients with histologically confirmed invasive BC stages I-III and IV, as well from 32 healthy Saudi Arabian women using a QIAamp® DNA Mini Kit. The isolated DNA was quantified using a Qubit™ dsDNA BR Assay Kit with a Qubit 2.0 Fluorometer. Ion semiconductor sequencing technology with an Ion S5 System and AmpliSeq™ Cancer Hotspot Panel v2 were utilized to analyze ~2,800 mutations described in the Catalogue of Somatic Mutations in Cancer from 50 oncogenes and tumor suppressor genes. Ion Reporter Software v.5.6 was used to evaluate the genomic alterations in all the samples after alignment to the hg19 human reference genome. The results showed that out of the 50 genes, 26 mutations, including 17 (65%) missense point mutations (single nucleotide variants), and 9 (35%) frameshift (insertion/deletion) mutations, were identified in 11 genes across the cohort in 61 samples (95%). Mutations were predominantly focused on two genes, PIK3CA and TP53, in the BC genomes of the sample set. PIK3CA mutation, c.1173A>G located in exon 9, was identified in 15 patients (46.9%). The TP53 mutations detected were a missense mutation (c.215C>G) in 26 patients (86.70%) and 1 frameshift mutation (c.215_216insG) in 1 patient (3.33%), located within exon 3 and 5, respectively. This study revealed specific mutation profiles for every BC patient, Thus, the results showed that Ion Torrent DNA Sequencing technology may be a possible diagnostic and prognostic method for developing personalized therapy based on the patient's individual BC genome.

Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma.

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

A Comparison of Five Illumina, Ion Torrent, and Nanopore Sequencing Technology-Based Approaches for Whole Genome Sequencing of Sars-Cov-2

A Comparison of Five Illumina, Ion Torrent, and Nanopore Sequencing Technology-Based Approaches for Whole Genome Sequencing of Sars-Cov-2

Comparing Published Gut Microbiome Taxonomic Data Across Multinational Studies.

Nurse researchers are well poised to study the connection of the microbiome to health and disease. Evaluating published microbiome results can assist with study design and hypothesis generation. This article aims to present and define important analysis considerations in microbiome study planning and to identify genera shared across studies despite methodological differences. This methods article will highlight a workflow that the nurse scientist can use to combine and evaluate taxonomy tables for microbiome study or research proposal planning. We compiled taxonomy tables from 13 published gut microbiome studies that had used Ion Torrent sequencing technology. We searched for studies that had amplified multiple hypervariable (V) regions of the 16S rRNA gene when sequencing the bacteria from healthy gut samples. We obtained 15 taxonomy tables from the 13 studies, comprised of samples from four continents and eight V regions. Methodology among studies was highly variable, including differences in V regions amplified, geographic location, and population demographics. Nevertheless, of the 354 total genera identified from the 15 data sets, 25 were shared in all V regions and the four continents. When relative abundance differences across the V regions were compared, Dorea and Roseburia were statistically different. Taxonomy tables from Asian subjects had increased average abundances of Prevotella and lowered abundances of Bacteroides compared with the European, North American, and South American study subjects. Evaluating taxonomy tables from previously published literature is essential for study planning. The genera found from different V regions and continents highlight geography and V region as important variables to consider in microbiome study design. The 25 shared genera across the various studies may represent genera commonly found in healthy gut microbiomes. Understanding the factors that may affect the results from a variety of microbiome studies will allow nurse scientists to plan research proposals in an informed manner. This work presents a valuable framework for future cross-study comparisons conducted across the globe.

PipeIT2: Somatic Variant Calling Workflow for Ion Torrent Sequencing Data.

The detection of somatic genetic variants forms the cornerstone of precision oncology. Molecular diagnostics laboratories require economical and rapid protocols to generate accurate variant calls for clinical purposes. The Ion Torrent sequencing platform is frequently used but lacks robust analysis workflows tailored to the needs of custom sequencing panels. We present PipeIT2, a somatic variant calling workflow for both matched tumor-germline and tumor-only analyses. PipeIT2 is enclosed in a Singularity container to ensure reproducibility and ease of deployment.

Bacterial diversity of geochemically distinct hot springs located in Maharashtra, India

Bacterial diversity of four thermally different hot springs of Ratnagiri district, Maharashtra, India, was investigated using culture-dependent and culture-independent approaches. A total of 144 bacterial cultures were isolated and identified using MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and 16S rRNA gene sequencing. Culture-independent analysis by Ion Torrent sequencing targeting the V3 region of the 16S rRNA gene revealed the predominance of Firmicutes across all the hot springs, followed by Chloroflexi, Bacteroidetes, Cyanobacteria, Proteobacteria, Armatimonadetes, Actinobacteria, Nitrospirae, Acidobacteria, and Deinococcus-Thermus, with subtle differences in their abundance. At the lower taxonomic rank of genus, we noted the prevalence of Acinetobacter followed by Clostridium, Planomicrobium, Bacillus, Streptomyces, and Leptolyngbya. Metagenomics imputation using in silico approach revealed divergence in the metabolic capabilities of bacterial communities along the thermal gradient of host springs, with site TS (63°C) featuring the abundant functional gene families.

Forensic nanopore sequencing of microhaplotype markers using QitanTech’s QNome

In recent years, extraordinary progress has been made in genome sequencing technologies, which has led to a decrease in cost and an increase in the diversity of sequenced genomes. Nanopore sequencing is one of the latest genome sequencing technologies. It aims to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions, and provides a new approach for forensic genetics to detect longer markers in real time. To date, multiple studies have been conducted to sequence forensic markers using MinION from Oxford Nanopore Technologies (ONT), and the results indicate that nanopore sequencing holds promise for forensic applications. Qitan Technology (QitanTech) recently launched its first commercial nanopore genome sequencer, QNome. It could achieve a read length of more than 150 kbp, and could generate approximately 500 Mb of data in 8 h. In this pilot study, we explored and validated this alternative nanopore sequencing device for microhaplotype (MH) profiling using a custom set of 15 MH loci. Seventy single-contributor samples were divided into 7 batches, each of which included 10 samples and control DNA 9947A and was sequenced by QNome. MH genotypes generated from QNome were compared to those from Ion Torrent sequencing (Ion S5XL system) to evaluate the accuracy and stability. Twelve samples randomly selected from the last three batches and Control DNA 9947A were also subjected to ONT MinION sequencing (with R9.4 flow cell) for parallel comparison. Based on MHtyper, a bioinformatics workflow developed for automated MH designation, all MH loci can be genotyped and reliably phased using the QNome data, with an overall accuracy of 99.83% (4 errors among 2310 genotypes). Three occurred near or in the region of homopolymer sequences, and one existed within 50 bp of the start of the sequencing reaction. In the last 15 samples (12 individual samples and 3 replicates of control DNA 9947A), two SNPs located at 4-mer homopolymers failed to obtain reliable genotypes on the MinION data. This study shows the potential of state-of-the-art nanopore sequencing methods to analyze forensic MH markers. Given the rapid pace of change, sporadic and nonrepetitive errors presented in this study are expected to be resolved by further developments of nanopore technologies and analysis tools.

Molecular profiling of breast and lung cancer in women with HIV reveals high tumor mutational burden.

This study compared the mutation profile and tumor mutational burden (TMB) in women with HIV (WWH) diagnosed with lung adenocarcinoma (n = 8) or breast ductal neoplasm (n = 13) who were enrolled into the Women's Interagency HIV Study (WIHS). Previous studies tended to focus on single institutions based on sample availability. This study is based on a representative, multicenter cohort that represents the racial and ethnic composition of women with HIV in the United States. The study sequenced the complete human exome of n = 26 cancer samples from HIV-positive women, using Ion torrent next-generation sequencing. The study cohort was compared with a HIV-negative cohort obtained from the Genomic Data Commons Data Portal of the NCI. There were no differences in known cancer mutations between breast cancer and lung cancer that developed in WWH and those that developed in HIV-negative (HIV-) women; however, WWH presented a significantly higher TMB in comparison to HIV- patients. Seventy-five percent of lung cancers and 61% of breast cancers were defined as TMB-high (more than 10 mutation/mb of DNA). This study affirms the recommendation that WWH be included in clinical trials of novel treatments for these cancers. Although these data are preliminary, the high TMB in WLHV suggests, paradoxically, that this immune challenged population may benefit greatly from immune checkpoint inhibitor therapies.

511. Treatment with Molnupiravir in the MOVe-In and MOVe-Out Clinical Trials Results in an Increase in Transition Mutations Across the SARS-CoV-2 Genome

BackgroundMolnupiravir (MOV), (MK-4482, EIDD-2801) is being clinically developed for the treatment of COVID-19 disease caused by SARS-CoV-2. MOV is the orally administered 5′-isobutyrate prodrug of the active, antiviral ribonucleoside analogue, N-hydroxycytidine (NHC, EIDD-1931) which inhibits viral replication by induction of mutations in the viral genome, leading to viral error catastrophe. In 2 clinical studies, hospitalized (MOVe-In) and non-hospitalized (MOVe-Out) participants were treated for 5 days with MOV and followed up to Day 29. Viral RNA isolated from nasal swab samples were sequenced to determine the rate, distribution and type of viral mutations observed after MOV treatment. MethodsRNA isolated from nasopharangeal swab samples collected during study conduct was quantified by RT-PCR. Samples containing >22,000 copies/mL of RNA underwent complete genome NGS using the Ion AmpliSeq SARS-CoV-2 research panel and Ion Torrent sequencing. Mutation rates were calculated by determining the number of nucleotide changes observed across the entire genome at Day 3 and/or Day 5 compared to baseline. ResultsCombined data from both studies showed an increase of ~2-4 fold in the viral mutation rate post-baseline in MOV treated compared with placebo. Mutations were distributed across the entire genome with only a minority being observed in more than one sample. The most frequent mutations were transitions of C to U observed in the highest MOV dose group (800 mg/BID).ConclusionConsistent with the proposed mechanism of action of MOV, an increase in the rate of transition mutations in the virus was observed in post-baseline nasal swab samples from participants treated with MOV compared with placebo. Disclosures Julie Strizki, PhD, Merck & Co., Inc. (Employee, Shareholder) Jay Grobler, PhD, Merck & Co., Inc. (Employee, Shareholder) Ying Zhang, PhD, Merck & Co., Inc. (Employee, Shareholder) Jiejun Du, PhD, Merck & Co., Inc. (Employee, Shareholder) Shunbing Zhao, PhD, Merck & Co., Inc. (Employee, Shareholder) Diane Levitan, PhD, Merck & Co., Inc. (Employee, Shareholder) Alex Therien, PhD, Merck & Co., Inc. (Employee, Shareholder) Joan R. Butterton, MD, Merck Sharp & Dohme Corp. (Employee, Shareholder) Nicholas Murgolo, PhD, Merck & Co., Inc. (Employee, Shareholder)

543. Molnupiravir Maintains Antiviral Activity Against SARS-CoV-2 Variants In Vitro and in Early Clinical Studies

BackgroundMolnupiravir (MOV, MK-4482, EIDD-2801) is an orally administered prodrug of N-hydroxycytidine (NHC, EIDD-1931), a nucleoside with broad antiviral activity against a range of RNA viruses. MOV acts by driving viral error catastrophe following its incorporation by the viral RdRp into the viral genome. Given its mechanism of action, MOV activity should not be affected by substitutions in the spike protein present in SARS-CoV-2 variants of concern which impact efficacy of therapeutic neutralizing antibodies and vaccine induced immunity. We characterized MOV activity against variants by assessing antiviral activity in vitro and virologic response from the Phase 2/3 clinical trials (MOVe-In, MOVe-Out) for treatment of COVID-19. MethodsMOV activity against several SARS-CoV-2 variants, was evaluated in an in vitro infection assay. Antiviral potency of NHC (IC50) was determined in Vero E6 cells infected with virus at MOI ~0.1 by monitoring CPE. Longitudinal SARS-CoV-2 RNA viral load measures in participants enrolled in MOVe-In and MOVe-Out were analyzed based on SARS-CoV-2 genotype. Sequences of SARS-CoV-2 from study participants were amplified from nasal swabs by PCR and NGS was performed on samples with viral genome RNA of >22,000 copies/ml amplified by primers covering full length genome with Ion Torrent sequencing to identify clades represented in trial participants. SARS-CoV-2 clades were assigned using clade.nextstrain.org. ResultsIn vitro, NHC was equally effective against SARS-CoV-2 variants B.1.1.7 (20I), B.1351 (20H), and P1 (20J), compared with the original WA1 (19B) isolate. In clinical trials, no discernable difference was observed in magnitude of viral response measured by change from baseline in RNA titer over time across all clades represented including 20A through 20E and 20G to 20I. No participants at the time of the study presented with 20F, 20J, or 21A.ConclusionDistribution of clades in participants in MOVe-In and MOVe-Out was representative of those circulating globally at the time of collection (Oct 2020 – Jan 2021). Both in vitro and clinical data suggest that spike protein substitutions do not impact antiviral activity of MOV and suggest its potential use for the treatment of SARS-CoV-2 variants.Disclosures Jay Grobler, PhD, Merck & Co., Inc. (Employee, Shareholder) Julie Strizki, PhD, Merck & Co., Inc. (Employee, Shareholder) Nicholas Murgolo, PhD, Merck & Co., Inc. (Employee, Shareholder) Wei Gao, PhD, Merck & Co., Inc. (Employee, Shareholder) Youfang Cao, PhD, Merck & Co. (Employee) Ying Zhang, PhD, Merck & Co., Inc. (Employee, Shareholder) Jiejun Du, PhD, Merck & Co., Inc. (Employee, Shareholder) Manoj Nair, PhD, Merck & Co., Inc. (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support) Yaoxing Huang, PhD, Merck & Co., Inc. (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support) Yang Luo, PhD, Merck & Co., Inc. (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support) Daria Hazuda, PhD, Merck & Co., Inc. (Employee, Shareholder) David D. Ho, MD, Merck & Co., Inc. (Grant/Research Support, Scientific Research Study Investigator, Research Grant or Support) David D. Ho, MD, Brii Biosciences (Individual(s) Involved: Self): Consultant; Merck (Individual(s) Involved: Self): Research Grant or Support; RenBio (Individual(s) Involved: Self): Consultant, Founder, Other Financial or Material Support, Shareholder; WuXi Biologics (Individual(s) Involved: Self): Consultant

From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel.

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels of variants found with both pipelines.