368. Performance Characteristics of Sequencing Assays for Identification of the SARS-CoV-2 Viral Genome

Abstract Background As the SARS-CoV-2 (SCV-2) virus evolves, diagnostics and vaccines against novel strains rely on viral genome sequencing. Researchers have gravitated towards the cost-effective and highly sensitive amplicon-based (e.g. ARTIC) and hybrid capture sequencing (e.g. SARS-CoV-2 NGS Assay) to selectively target the SCV-2 genome. We provide an in silico model to compare these 2 technologies and present data on the high scalability of the Research Use Only (RUO) workflow of the SARS-CoV-2 NGS Assay. Methods In silico work included alignments of 383,656 high-quality genome sequences belonging to variant of concern (VOC) or variant of interest (VOI) isolates (GISAID). We profiled mismatches and sequencing dropouts using the ARTIC V3 primers, SARS-CoV-2 NGS Assay probes (Twist Bioscience) and 11 synthesized viral sequences containing mutations and compared the performance of these assays using clinical samples. Further, the miniaturized hybrid capture workflow was optimized and evaluated to support high-throughput (384-plex). The sequencing data was processed by COVID-DX software. Results We detected 101,432 viruses (27%) with > = 1 mismatch in the last 6 base pairs of the 3’ end of ARTIC primers; of these, 413 had > = 2 mismatches in one primer. In contrast, only 38 viruses (0.01%) had enough mutations ( > = 10) in a hybrid capture probe to have a similar effect on coverage. We observed that mutations in ARTIC primers led to complete dropout of the amplicon for 4/11 isolates and diminished coverage in additional 4. Twist probes showed uniform coverage throughout with little to no dropouts. Both assays detected a wide range of variants (~99.9% coverage at 5X depth) in clinical samples (CT value < 30) collected in NY (Spring 2020-Spring 2021). The distribution of the number of reads and on target rates were more uniform among specimens within amplicon-based sequencing. However, uneven genome coverage and primer dropouts, some in the spike protein, were observed on VOC/VOI and other isolates highlighting limitations of an amplicon-based approach. Conclusion The RUO workflow of the SARS-CoV-2 NGS Assay is a comprehensive and scalable sequencing tool for variant profiling, yields more consistent coverage and smaller dropout rate compared to ARTIC (0.05% vs. 7.7%). Disclosures Danny Antaki, PhD, Twist Bioscience (Employee, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee, Shareholder) Esteban Toro, PhD, Twist Bioscience (Employee) Bryan Höglund, BS, Twist Bioscience (Employee, Shareholder) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Joseph Barrows, MS, Biotia (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder)

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423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.617 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S278-S279

Author(s):

Dorottya Nagy-Szakal ◽

Mara Couto-Rodriguez ◽

Joseph Barrows ◽

Heather L Wells ◽

Marilyne Debieu ◽

...

Keyword(s):

Genetic Variants ◽

Viral Genome ◽

Board Member ◽

Limit Of Detection ◽

Clinical Samples ◽

Analysis Tool ◽

Viral Titer ◽

Target Enrichment ◽

Library Preparation ◽

Software Analysis

Abstract Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

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Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater

International Journal of Molecular Sciences ◽

10.3390/ijms22168498 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8498

Author(s):

Margaritis Avgeris ◽

Panagiotis G. Adamopoulos ◽

Aikaterini Galani ◽

Marieta Xagorari ◽

Dimitrios Gourgiotis ◽

...

Keyword(s):

Real Time ◽

Nested Pcr ◽

Mutational Analysis ◽

National Level ◽

Rna Stability ◽

3D Structure ◽

Cost Effective ◽

Clinical Samples ◽

Sequencing Data ◽

Alpha Variant

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%–60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients’ sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

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Biomarker-driven indication selection in JTX-2011 ICONIC clinical trial.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.11602 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 11602-11602

Author(s):

Heather Anne Hirsch ◽

Jason Reeves ◽

Tong Zi ◽

Amit Deshpande ◽

Guang Yang ◽

...

Keyword(s):

Solid Tumors ◽

In Silico ◽

Immune Cell ◽

Ex Vivo ◽

Single Agent ◽

Predictive Biomarkers ◽

Clinical Samples ◽

Integrated Analysis ◽

T Effector Cells ◽

Wide Range

11602 Background: ICOS (Inducible T cell CO-Stimulator) is a co-stimulatory molecule expressed primarily on T lymphocytes. Clinical and preclinical data suggest that ICOS mediates anti-CTLA-4 driven anti-tumor responses. JTX-2011 is an ICOS agonist antibody in clinical development in advanced solid tumors (ICONIC trial). JTX-2011 is designed to generate an anti-tumor immune response via stimulation of T effector cells and preferential reduction of intra-tumoral T regulatory cells. Single agent preclinical efficacy correlates with the percentage of ICOS-expressing T cells within the tumor. We report indication selection and patient enrichment strategy for ICONIC using in silico and IHC analysis and assessment of potential predictive biomarkers for JTX-2011 using ex vivo tumor histoculture. Methods: Integrated analysis was performed from the TCGA for ICOS expression in histologic and molecularly defined tumors and immune cell signatures. ICOS expression was analyzed by IHC in a subset of indications based on in silico analysis. ICOS expression on intra-tumoral Tregs and PD-L1 were analyzed in a cohort of 126 head and neck squamous cell carcinomas (HNSCC). Ex vivo histoculture assays of human HNSCC was treated with JTX-2011 and assessed for IFNg gene signature induction. Results: ICOS mRNA expression was analyzed in ~10,000 solid tumors samples across ~30 indications. ICOS expression in key indications was confirmed using IHC. Based on frequency of high ICOS expression, non-small cell lung cancer, HNSCC, triple negative breast carcinoma, gastric cancer, and melanoma were selected as indications for ICONIC. Results were confirmed in clinical samples using multiplex immunofluorescence and ICOS IHC. A wide range of ICOS expression was observed suggesting that identification of an ICOS “high” group may enrich for patients likely to benefit from ICOS agonist therapy. In ex vivo histoculture assays of human HNSCC tumors treated with JTX-2011, ICOS IHC and ICOS RNA gene signatures correlated to response endpoints. Comparison of ICOS and PDL1 expression identified subsets of tumors in multiple indications with high ICOS but low PDL1 expression. Conclusions: These data support prioritization of specific tumor types the ICONIC trial.

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Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

10.1101/2021.11.10.467884 ◽

2021 ◽

Author(s):

Isabel S Naarmann-de Vries ◽

Christiane Zorbas ◽

Amina Lemsara ◽

Maja Bencun ◽

Sarah Schudy ◽

...

Keyword(s):

Rna Sequencing ◽

Clinical Samples ◽

Sequencing Data ◽

Rna Modifications ◽

Flow Cells ◽

Wide Range ◽

Catalytically Active ◽

Induced Pluripotent ◽

First Time

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridine, 2-O-methylation of ribose and acetylation of cytidine. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

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Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution

Current Issues in Molecular Biology ◽

10.3390/cimb43030134 ◽

2021 ◽

Vol 43 (3) ◽

pp. 1937-1949

Author(s):

Laura A. E. Van Poelvoorde ◽

Mathieu Gand ◽

Marie-Alice Fraiture ◽

Sigrid C. J. De Keersmaecker ◽

Bavo Verhaegen ◽

...

Keyword(s):

In Silico ◽

Virus Evolution ◽

Clinical Samples ◽

Whole Genome Sequencing Data ◽

Preliminary Evaluation ◽

Whole Genome ◽

Sequencing Data ◽

Method Performance ◽

The World ◽

Pcr Assays

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

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Inference of Chromosome-Length Haplotypes Using Genomic Data of Three or a Few More Single Gametes

Molecular Biology and Evolution ◽

10.1093/molbev/msaa176 ◽

2020 ◽

Vol 37 (12) ◽

pp. 3684-3698 ◽

Cited By ~ 1

Author(s):

Ruidong Li ◽

Han Qu ◽

Jinfeng Chen ◽

Shibo Wang ◽

John M Chater ◽

...

Keyword(s):

Large Scale ◽

Low Cost ◽

Genomic Data ◽

Cost Effective ◽

R Package ◽

Chromosome Length ◽

Sequencing Data ◽

Experimental Phasing ◽

Wide Range ◽

Haplotype Data

Abstract Compared with genomic data of individual markers, haplotype data provide higher resolution for DNA variants, advancing our knowledge in genetics and evolution. Although many computational and experimental phasing methods have been developed for analyzing diploid genomes, it remains challenging to reconstruct chromosome-scale haplotypes at low cost, which constrains the utility of this valuable genetic resource. Gamete cells, the natural packaging of haploid complements, are ideal materials for phasing entire chromosomes because the majority of the haplotypic allele combinations has been preserved. Therefore, compared with the current diploid-based phasing methods, using haploid genomic data of single gametes may substantially reduce the complexity in inferring the donor’s chromosomal haplotypes. In this study, we developed the first easy-to-use R package, Hapi, for inferring chromosome-length haplotypes of individual diploid genomes with only a few gametes. Hapi outperformed other phasing methods when analyzing both simulated and real single gamete cell sequencing data sets. The results also suggested that chromosome-scale haplotypes may be inferred by using as few as three gametes, which has pushed the boundary to its possible limit. The single gamete cell sequencing technology allied with the cost-effective Hapi method will make large-scale haplotype-based genetic studies feasible and affordable, promoting the use of haplotype data in a wide range of research.

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Genetic Sex Validation for Sample Tracking in Clinical Testing

10.1101/2021.12.13.470986 ◽

2021 ◽

Author(s):

Jianhong Hu ◽

Viktoriya Korchina ◽

Hana Zouk ◽

Maegan V. Harden ◽

David Murdock ◽

...

Keyword(s):

Quality Control ◽

Dna Sequencing ◽

Control Measure ◽

Cost Effective ◽

Marrow Transplant ◽

Phase Iii ◽

Clinical Samples ◽

Clinical Testing ◽

Sequencing Data ◽

Sample Collection

Background: Next generation DNA sequencing (NGS) has been rapidly adopted by clinical testing laboratories for detection of germline and somatic genetic variants. The complexity of sample processing in a clinical DNA sequencing laboratory creates multiple opportunities for sample identification errors, demanding stringent quality control procedures. Methods: We utilized DNA genotyping via a 96-SNP PCR panel applied at sample acquisition in comparison to the final sequence, for tracking of sample identity throughout the sequencing pipeline. The 96-SNP PCR panel's inclusion of sex SNPs also provides a mechanism for a genotype-based comparison to recorded sex at sample collection for identification. This approach was implemented in the clinical genomic testing pathways, in the multi-center Electronic Medical Records and Genomics (eMERGE) Phase III program. Results: We identified 110 inconsistencies from 25,015 (0.44%) clinical samples, when comparing the 96-SNP PCR panel data to the test requisition-provided sex. The 96-SNP PCR panel genetic sex predictions were confirmed using additional SNP sites in the sequencing data or high-density hybridization-based genotyping arrays. Results identified clerical errors, samples from transgender participants and stem cell or bone marrow transplant patients and undetermined sample mix-ups. Conclusion: The 96-SNP PCR panel provides a cost-effective, robust tool for tracking samples within DNA sequencing laboratories, while the ability to predict sex from genotyping data provides an additional quality control measure for all procedures, beginning with sample collections. While not sufficient to detect all sample mix-ups, the inclusion of genetic versus reported sex matching can give estimates of the rate of errors in sample collection systems.

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Neuroblastoma Risk Assessment and Treatment Stratification with Hybrid Capture-Based Panel Sequencing

Journal of Personalized Medicine ◽

10.3390/jpm11080691 ◽

2021 ◽

Vol 11 (8) ◽

pp. 691

Author(s):

Annabell Szymansky ◽

Louisa-Marie Kruetzfeldt ◽

Lukas C. Heukamp ◽

Falk Hertwig ◽

Jessica Theissen ◽

...

Keyword(s):

Genome Sequencing ◽

Neuroblastoma Cell ◽

Tumor Biology ◽

Clinical Samples ◽

Risk Markers ◽

Sequencing Data ◽

High Coverage ◽

Routine Diagnostics ◽

Hybrid Capture ◽

Panel Sequencing

For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our “neuroblastoma hybrid capture sequencing panel” (NB-HCSP) assay employs a technology for the high-coverage sequencing (>1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.

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Identifying differentially methylated sites in samples with varying tumor purity

10.1101/248781 ◽

2018 ◽

Author(s):

Antti Häkkinen ◽

Amjad Alkodsi ◽

Chiara Facciotto ◽

Kaiyang Zhang ◽

Katja Kaipio ◽

...

Keyword(s):

Normal Cell ◽

Computational Method ◽

Clinical Samples ◽

Whole Genome Sequencing Data ◽

Sequencing Data ◽

Cell Control ◽

Tumor Purity ◽

Wide Range ◽

Cancer Types ◽

Control Samples

AbstractDNA methylation aberrations are common in many cancer types. A major challenge hindering comparison of patient-derived samples is that they comprise of heterogeneous collection of cancer and microenvironment cells. We present a computational method that allows comparing cancer methylomes in two or more heterogeneous tumor samples featuring differing, unknown fraction of cancer cells. The method is unique in that it allows comparison also in the absence of normal cell control samples and without prior tumor purity estimates, as these are often unavailable or unreliable in clinical samples. We use simulations and next-generation methylome, RNA, and whole-genome sequencing data from two cancer types to demonstrate that the method is accurate and outperforms alternatives. The results show that our method adapts well to various cancer types and to a wide range of tumor content, and works robustly without a control or with controls derived from various sources. The method is freely available at https://bitbucket.org/anthakki/dmml.

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2D Color-Intensity Representation of Ultrasonic Thickness Gauge Measurements

Materials Evaluation ◽

10.32548/2020.me-04177 ◽

2020 ◽

pp. 1192-1198

Author(s):

M.S. Mohammad ◽

Tibebe Tesfaye ◽

Kim Ki-Seong

Keyword(s):

Cost Effective ◽

The Other ◽

Thickness Gauge ◽

Color Intensity ◽

Digital Readout ◽

Flat Surfaces ◽

Video Cameras ◽

Wide Range ◽

Cost Effective Alternative ◽

Ultrasonic Thickness

Ultrasonic thickness gauges are easy to operate and reliable, and can be used to measure a wide range of thicknesses and inspect all engineering materials. Supplementing the simple ultrasonic thickness gauges that present results in either a digital readout or as an A-scan with systems that enable correlating the measured values to their positions on the inspected surface to produce a two-dimensional (2D) thickness representation can extend their benefits and provide a cost-effective alternative to expensive advanced C-scan machines. In previous work, the authors introduced a system for the positioning and mapping of the values measured by the ultrasonic thickness gauges and flaw detectors (Tesfaye et al. 2019). The system is an alternative to the systems that use mechanical scanners, encoders, and sophisticated UT machines. It used a camera to record the probe’s movement and a projected laser grid obtained by a laser pattern generator to locate the probe on the inspected surface. In this paper, a novel system is proposed to be applied to flat surfaces, in addition to overcoming the other limitations posed due to the use of the laser projection. The proposed system uses two video cameras, one to monitor the probe’s movement on the inspected surface and the other to capture the corresponding digital readout of the thickness gauge. The acquired images of the probe’s position and thickness gauge readout are processed to plot the measured data in a 2D color-coded map. The system is meant to be simpler and more effective than the previous development.

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