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

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)

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

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S286-S287
Author(s):  
Danny Antaki ◽  
Mara Couto-Rodriguez ◽  
Tong Liu ◽  
Kristin Butcher ◽  
Esteban Toro ◽  
...  
Keyword(s):  
In Silico ◽  
Viral Genome ◽  
Board Member ◽  
Panel Member ◽  
Cost Effective ◽  
Dropout Rate ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Hybrid Capture ◽  
Wide Range

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)

scholarly journals Library Preparation Based on Transposase Assisted RNA/DNA Hybrid Co-Tagmentation for Next-Generation Sequencing of Human Noroviruses

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 65
Author(s):  
Zilei Zhang ◽  
Danlei Liu ◽  
Dapeng Wang ◽  
Qingping Wu
Keyword(s):  
Next Generation Sequencing ◽  
Viral Genome ◽  
Viral Metagenomics ◽  
Clinical Samples ◽  
Whole Genome ◽  
Library Preparation ◽  
Next Generation ◽  
Essential Information ◽  
Human Noroviruses ◽  
Generation Sequencing

Human noroviruses (HuNoVs) are one of the leading causes of foodborne illnesses globally. The viral genome is the most essential information for viral source tracing and viral transmission pattern monitoring. However, whole genome sequencing of HuNoVs is still challenging due to the sequence heterogeneity among different genotypes and low titer in samples. To address this need, in this study, the Transposase assisted RNA/DNA hybrid Co-tagmentation (TRACE-seq) method was established for next generation sequencing library preparation of HuNoVs. Our data demonstrated that almost the whole HuNoVs genome (>7 kb) could be obtained from all of the 11 clinical samples tested. Twelve genotypes including GI.3, GI.4, GI.5, GI.8, GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, and GII.21 were involved. Compared with the traditional method for viral metagenomics library preparation, optimized TRACE-seq greatly reduced the interference from the host’s and bacterial RNAs. In addition, viral genome sequences can be assembled by using less raw data with sufficient depth along the whole genome. Therefore, for the high versatility and reliability, this method is promising for whole viral genome attainment. It is particularly applicable for the viruses with a low titer that are mixed with a complicated host background and are unable to be cultured in vitro, like the HuNoVs utilized in this study.

scholarly journals Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexia Bordigoni ◽  
Anne Motte ◽  
Hervé Tissot-Dupont ◽  
Philippe Colson ◽  
Christelle Desnues
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Papillomaviruses ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Gene Encoding ◽  
Hpv Dna ◽  
E6 And E7 ◽  
High Risk Hpv ◽  
Development And Validation

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

scholarly journals Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anita G. Amin ◽  
Prithwiraj De ◽  
Barbara Graham ◽  
Roger I. Calderon ◽  
Molly F. Franke ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sample Pretreatment ◽  
Positive Association ◽  
Proteinase K ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Smear Negative ◽  
Tuberculostearic Acid ◽  
Negative Sputum ◽  
Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

scholarly journals NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR

2020 ◽  
Author(s):  
Fangyan Yu ◽  
Ka Wai Leong ◽  
Alexander Makrigiorgos ◽  
Viktor A Adalsteinsson ◽  
Ioannis Ladas ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Analysis Tool ◽  
Circulating Dna ◽  
Liquid Biopsies ◽  
Software Analysis ◽  
Multiplexed Pcr ◽  
Human Genomes ◽  
Custom Made ◽  
Alu Element ◽  
Next Generation Sequencing Ngs

Abstract Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80–150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05–1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.

scholarly journals RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 139
Author(s):  
Caterina De Luca ◽  
Francesco Pepe ◽  
Antonino Iaccarino ◽  
Pasquale Pisapia ◽  
Luisella Righi ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Limit Of Detection ◽  
Predictive Biomarkers ◽  
Small Cell ◽  
Clinical Samples ◽  
Gene Fusions ◽  
Small Cell Lung ◽  
Relevant Gene

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades

2021 ◽  
Vol 7 (6) ◽  
pp. 433
Author(s):  
Ahmad Ibrahim ◽  
Lucie Peyclit ◽  
Rim Abdallah ◽  
Saber Khelaifia ◽  
Amanda Chamieh ◽  
...  
Keyword(s):  
Culture Medium ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Candida Auris ◽  
Phenotypic Profile ◽  
Stool Samples ◽  
Selective Culture Medium

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

scholarly journals Sandwich-Type DNA Micro-Optode Based on Gold–Latex Spheres Label for Reflectance Dengue Virus Detection

Sensors ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 1820
Author(s):  
Jeningsih ◽  
Ling Ling Tan ◽  
Alizar Ulianas ◽  
Lee Yook Heng ◽  
Nur-Fadhilah Mazlan ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Limit Of Detection ◽  
Virus Detection ◽  
Average Diameter ◽  
Optical Biosensor ◽  
Clinical Samples ◽  
Sandwich Hybridization ◽  
Latex Spheres ◽  
Sandwich Type ◽  
Thiol Binding

A DNA micro-optode for dengue virus detection was developed based on the sandwich hybridization strategy of DNAs on succinimide-functionalized poly(n-butyl acrylate) (poly(nBA-NAS)) microspheres. Gold nanoparticles (AuNPs) with an average diameter of ~20 nm were synthesized using a centrifugation-based method and adsorbed on the submicrometer-sized polyelectrolyte-coated poly(styrene-co-acrylic acid) (PSA) latex particles via an electrostatic method. The AuNP–latex spheres were attached to the thiolated reporter probe (rDNA) by Au–thiol binding to functionalize as an optical gold–latex–rDNA label. The one-step sandwich hybridization recognition involved a pair of a DNA probe, i.e., capture probe (pDNA), and AuNP–PSA reporter label that flanked the target DNA (complementary DNA (cDNA)). The concentration of dengue virus cDNA was optically transduced by immobilized AuNP–PSA–rDNA conjugates as the DNA micro-optode exhibited a violet hue upon the DNA sandwich hybridization reaction, which could be monitored by a fiber-optic reflectance spectrophotometer at 637 nm. The optical genosensor showed a linear reflectance response over a wide cDNA concentration range from 1.0 × 10−21 M to 1.0 × 10−12 M cDNA (R2 = 0.9807) with a limit of detection (LOD) of 1 × 10−29 M. The DNA biosensor was reusable for three consecutive applications after regeneration with mild sodium hydroxide. The sandwich-type optical biosensor was well validated with a molecular reverse transcription polymerase chain reaction (RT-PCR) technique for screening of dengue virus in clinical samples, e.g., serum, urine, and saliva from dengue virus-infected patients under informed consent.

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