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

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.

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Significant Advances in the Management of Patients with Hematological Disorders, New Challenges and Immense Responsibilities

Journal of Clinical Cases & Reports ◽

10.46619/joccr.2018.1-1003 ◽

2018 ◽

Vol 1 (1) ◽

pp. 8-9

Author(s):

Laribi Kamel ◽

◽

Baugier de Materre Alix ◽

Keyword(s):

Flow Cytometry ◽

Residual Disease ◽

New Drugs ◽

Circulating Dna ◽

Liquid Biopsies ◽

Hematological Disorders ◽

New Challenges ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The recent years have seen an acceleration in understanding the physio-pathological mechanisms of many diseases, as well as improvement in their follow-up with more and more powerful tools like flow cytometry, quantitative PCR and more recently the next generation sequencing (NGS), as well as the follow-up of residual disease on liquid biopsies (circulating DNA) but especially the discovery of many new drugs that significantly improved patient’s survival.

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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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A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00586-y ◽

2021 ◽

Author(s):

Seyoung Mun ◽

Songmi Kim ◽

Wooseok Lee ◽

Keunsoo Kang ◽

Thomas J. Meyer ◽

...

Keyword(s):

Genome Sequencing ◽

Genome Assembly ◽

De Novo ◽

Personal Genome ◽

Human Populations ◽

Whole Genome ◽

Structural Variations ◽

Insert Size ◽

Human Genomes ◽

Next Generation Sequencing Ngs

AbstractAdvances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

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Measuring myocardial extracellular volume of the right ventricle in patients with congenital heart disease

Scientific Reports ◽

10.1038/s41598-021-81440-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nadya Al-Wakeel-Marquard ◽

Tiago Ferreira da Silva ◽

Sarah Jeuthe ◽

Sanaz Rastin ◽

Frédéric Muench ◽

...

Keyword(s):

Congenital Heart Disease ◽

Heart Disease ◽

Image Quality ◽

Right Ventricle ◽

Wall Thickness ◽

Congenital Heart ◽

Extracellular Volume ◽

Analysis Tool ◽

Custom Made ◽

The Right

AbstractThe right ventricle´s (RV) characteristics—thin walls and trabeculation—make it challenging to evaluate extracellular volume (ECV). We aimed to assess the feasibility of RV ECV measurements in congenital heart disease (CHD), and to introduce a novel ECV analysis tool. Patients (n = 39) and healthy controls (n = 17) underwent cardiovascular magnetic resonance T1 mapping in midventricular short axis (SAX) and transverse orientation (TRANS). Regions of interest (ROIs) were evaluated with regard to image quality and maximum RV wall thickness per ROI in pixels. ECV from plane ROIs was compared with values obtained with a custom-made tool that derives the mean T1 values from a “line of interest” (LOI) centered in the RV wall. In CHD, average image quality was good (no artifacts in the RV, good contrast between blood/myocardium), and RV wall thickness was 1–2 pixels. RV ECV was not quantifiable in 4/39 patients due to insufficient contrast or wall thickness < 1 pixel. RV myocardium tended to be more clearly delineated in SAX than TRANS. ECV from ROIs and corresponding LOIs correlated strongly in both directions (SAX/TRANS: r = 0.97/0.87, p < 0.001, respectively). In conclusion, RV ECV can be assessed if image quality allows sufficient distinction between myocardium and blood, and RV wall thickness per ROI is ≥ 1 pixel. T1 maps in SAX are recommended for RV ECV analysis. LOI application simplifies RV ECV measurements.

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R-Score: A New Parameter to Assess the Quality of Variants’ Calls Assessed by NGS Using Liquid Biopsies

Biology ◽

10.3390/biology10100954 ◽

2021 ◽

Vol 10 (10) ◽

pp. 954

Author(s):

Roberto Serna-Blasco ◽

Estela Sánchez-Herrero ◽

María Berrocal Renedo ◽

Silvia Calabuig-Fariñas ◽

Miguel Ángel Molina-Vila ◽

...

Keyword(s):

Egfr Mutation ◽

False Negative ◽

Digital Pcr ◽

Liquid Biopsies ◽

Molecular Alterations ◽

Significant Linear Correlation ◽

Molecular Landscape ◽

Nsclc Patients ◽

Next Generation Sequencing Ngs

Next-generation sequencing (NGS) has enabled a deeper knowledge of the molecular landscape in non-small cell lung cancer (NSCLC), identifying a growing number of targetable molecular alterations in key genes. However, NGS profiling of liquid biopsies risk for false positive and false negative calls and parameters assessing the quality of NGS calls remains lacking. In this study, we have evaluated the positive percent agreement (PPA) between NGS and digital PCR calls when assessing EGFR mutation status using 85 plasma samples from 82 EGFR-positive NSCLC patients. According to our data, variant allele fraction (VAF) was significantly lower in discordant calls and the median of the absolute values of all pairwise differences (MAPD) was significantly higher in discordant calls (p < 0.001 in both cases). Based on these results, we propose a new parameter that integrates both variables, named R-score. Next, we sought to evaluate the PPA for EGFR mutation calls between two independent NGS platforms using a subset of 40 samples from the same cohort. Remarkably, there was a significant linear correlation between the PPA and the R-score (r = 0.97; p < 0.001). Specifically, the PPA of samples with an R-score ≤ −1.25 was 95.83%, whereas PPA falls to 81.63% in samples with R-score ≤ 0.25. In conclusion, R-score significantly correlates with PPA and can assist laboratory medicine specialists and data scientists to select reliable variants detected by NGS.

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An Analysis Tool for the Installation of Submarine Cables in an S-Lay Configuration Including “In and Out of Water” Cable Segments

Journal of Marine Science and Engineering ◽

10.3390/jmse8010048 ◽

2020 ◽

Vol 8 (1) ◽

pp. 48 ◽

Cited By ~ 1

Author(s):

Vasileios A. Mamatsopoulos ◽

Constantine Michailides ◽

Efstathios E. Theotokoglou

Keyword(s):

Finite Element Analysis ◽

Finite Element ◽

Design Criterion ◽

Limiting Factors ◽

Radius Of Curvature ◽

Analysis Tool ◽

Analysis Software ◽

Element Analysis ◽

Analysis And Design ◽

Custom Made

Today, the offshore oil and gas and wind power industry is a heavily regulated segment, and current standards have established restrictions which yield a very limited weather window for submarine cable installations due to experience with cable failure in bad weather. There are two main limiting factors in current practice during cable installation of an S-lay configuration: the design criterion for the minimum allowable radius of curvature in the touch down point and the avoidance of axial compression in the touch down zone. Accurate assessment of the cable integrity during offshore installation has drawn great attention and is related to the existing available analysis and design tools. The main purpose of this paper is to develop and propose a quick and easy custom-made analysis tool, which is able to export similar results as sophisticated finite element analysis software. The developed tool utilizes analytical equations of a catenary-type submarine structure extended to account for varying cross-sections with different weights and/or stiffnesses, as is the real practice. A comparative study is presented in this paper to evaluate the significance for the modeling of the “out of water” cable segment required for accurate safety factor quantification during a laying operation. The efficiency and accuracy of the proposed tool are proven through a validation study comparing the results and the computational effort and time with commercial finite element analysis software. The analysis error in the case of not modeling the “out of water” cable part is significant, especially in shallow water areas, which proves the importance of using the proposed analysis tool.

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Estimation in the Field of Individual Perennial Ryegrass Plant Position and Dry Matter Production Using a Custom-Made High-Throughput Image Analysis Tool

Crop Science ◽

10.2135/cropsci2015.02.0125 ◽

2015 ◽

Vol 55 (6) ◽

pp. 2910-2917 ◽

Cited By ~ 5

Author(s):

Chris L. Hunt ◽

Chris S. Jones ◽

Michael J. Hickey ◽

John P. Koolaard ◽

John West ◽

...

Keyword(s):

Image Analysis ◽

High Throughput ◽

Perennial Ryegrass ◽

Dry Matter ◽

Dry Matter Production ◽

Analysis Tool ◽

Matter Production ◽

Custom Made

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High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies

Clinical Chemistry ◽

10.1093/clinchem/hvaa013 ◽

2020 ◽

Vol 66 (4) ◽

pp. 606-613 ◽

Cited By ~ 3

Author(s):

Amanda Bortolini Silveira ◽

François-Clément Bidard ◽

Amélie Kasperek ◽

Samia Melaabi ◽

Marie-Laure Tanguy ◽

...

Keyword(s):

Microsatellite Instability ◽

Large Scale ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Diagnostic Tools ◽

Tumor Biomarker ◽

Analytical Sensitivity ◽

Liquid Biopsies ◽

Tumor Dna ◽

Large Scale Screening

Abstract Background Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. Methods We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. Results The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. Conclusions This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

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