scholarly journals Sequencing‐based microsatellite instability testing using as few as six markers for high‐throughput clinical diagnostics

2019 ◽  
Vol 41 (1) ◽  
pp. 332-341 ◽  
Author(s):  
Richard Gallon ◽  
Harsh Sheth ◽  
Christine Hayes ◽  
Lisa Redford ◽  
Ghanim Alhilal ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
High Throughput ◽  
Clinical Diagnostics

scholarly journals Directed Evolution of Aptamer Discovery Technologies

2021 ◽  
Author(s):  
Diana Wu ◽  
Chelsea Gordon ◽  
John Shin ◽  
Michael Eisenstein ◽  
Hyongsok Tom Soh
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Click Reaction ◽  
Clinical Diagnostics ◽  
High Quality ◽  
Affinity Reagents ◽  
Wide Range ◽  
Target Binding ◽  
Modified Nucleobases ◽  
Parallel Fashion

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

scholarly journals Importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

2002 ◽  
Author(s):  
James F. Leary ◽  
Peter Szaniszlo ◽  
Tarl W. Prow ◽  
Lisa M. Reece ◽  
Nan Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Separation ◽  
Clinical Diagnostics

scholarly journals CNV Workshop: an integrated platform for high-throughput copy number variation discovery and clinical diagnostics

2010 ◽  
Vol 11 (1) ◽  
pp. 74 ◽  
Author(s):  
Xiaowu Gai ◽  
Juan C Perin ◽  
Kevin Murphy ◽  
Ryan O'Hara ◽  
Monica D'arcy ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
High Throughput ◽  
Copy Number ◽  
Clinical Diagnostics ◽  
Integrated Platform ◽  
Number Variation

scholarly journals Diagnostic high-throughput sequencing of 2,390 patients with bleeding, thrombotic and platelet disorders

2018 ◽  
Author(s):  
Kate Downes ◽  
Karyn Megy ◽  
Daniel Duarte ◽  
Minka Vries ◽  
Johanna Gebhart ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Clinical Phenotype ◽  
Careful Consideration ◽  
Clinical Diagnostics ◽  
Genomic Variation ◽  
Panel Test ◽  
Platelet Disorders ◽  
Normal Hemostasis ◽  
And Function

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multi-disciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2,390 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbour variants associated with rare bleeding, thrombotic or platelet disorders (BPD). The diagnostic rate was determined by the clinical phenotype, with an overall rate of 50.4% for all thrombotic, coagulation, platelet count and function disorder patients and a rate of 6.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 756 unique variants, including copy number and intronic variants, as Pathogenic, Likely Pathogenic or Variants of Uncertain Significance. Almost half (49.7%) of these variants are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BPD. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 897 index patients providing evidence that introducing a HTS genetic test for BPD patients is meeting an important unmet clinical need.

scholarly journals Orthogonal NGS for High Throughput Clinical Diagnostics

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Niru Chennagiri ◽  
Eric J. White ◽  
Alexander Frieden ◽  
Edgardo Lopez ◽  
Daniel S. Lieber ◽  
...  
Keyword(s):  
High Throughput ◽  
Clinical Diagnostics

scholarly journals Microsatellite Instability in Colorectal Cancer: Considerations for Molecular Diagnosis and High-Throughput Screening of Archival Tissues

2004 ◽  
Vol 50 (6) ◽  
pp. 1082-1086 ◽  
Author(s):  
Manuel Salto-Tellez ◽  
Soo Chin Lee ◽  
Lily L Chiu ◽  
Chi Kuen Lee ◽  
May Chin Yong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Molecular Diagnosis ◽  
High Throughput ◽  
High Throughput Screening

Cold-microwave enhanced enzyme-linked immunosorbent assays—A path to high-throughput clinical diagnostics

2014 ◽  
Vol 457 ◽  
pp. 65-73 ◽  
Author(s):  
Niels Grützner ◽  
Romy M. Heilmann ◽  
Jan S. Suchodolski ◽  
Jörg M. Steiner ◽  
Andreas Holzenburg
Keyword(s):  
High Throughput ◽  
Clinical Diagnostics ◽  
Immunosorbent Assays

A molecular inversion probe and sequencing-based microsatellite instability assay for high throughput cancer diagnostics and Lynch syndrome screening

2018 ◽  
Author(s):  
Richard Gallon ◽  
Christine Hayes ◽  
Lisa Redford ◽  
Ghanim Alhilal ◽  
Ottie O’Brien ◽  
...  
Keyword(s):  
Lynch Syndrome ◽  
Microsatellite Instability ◽  
High Throughput ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Reliable Classification ◽  
Analysis System ◽  
Template Dna

AbstractBackgroundClinical guidelines recommend microsatellite instability (MSI) and BRAF V600E testing of all colorectal cancers (CRCs) to screen for Lynch syndrome (LS), a hereditary predisposition to cancer. MSI is also associated with response to immunotherapy. However, uptake of MSI testing is poor and current assays are not suitable for high throughput diagnostics.We aimed to develop a cheap and scalable sequencing assay for MSI classification, which is robust to variables in clinical samples and simultaneously tests for BRAF V600E to streamline the LS screening pipeline.Methods24 short (7-12bp) microsatellites and the BRAF V600E locus were amplified in multiplex using single molecule molecular inversion probes (smMIPs) and sequenced using the Illumina MiSeq platform. Reads were aligned to reference genome hg19. An MSI classifier was trained from 98 CRCs and validated in 99 independent CRCs collected in pathology laboratories in Edinburgh, Spain and Newcastle.ResultsThe smMIP-based MSI assay has 100% accuracy for MSI status relative to MSI Analysis System (Promega). MSI classification is reproducible (100% concordance) and is robust to sample variables, detecting less than 5% MSI-high content in template DNA and giving reliable classification from sequencing only 75 DNA molecules per marker. BRAF V600E was detected with mutant allele frequencies down to 1.7%.ConclusionsOur short microsatellite, smMIP-based, MSI assay provides a cheap and fully automatable assessment of MSI status and BRAF mutation. It is readily scalable to high throughput cancer diagnostics, and is suitable both as a companion diagnostic for immunotherapy and for streamlined LS screening.

scholarly journals Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Elitza S. Theel ◽  
Julie Harring ◽  
Heather Hilgart ◽  
Dane Granger
Keyword(s):  
High Throughput ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Clinical Diagnostics ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Predictive Values ◽  
Serologic Tests ◽  
Convalescent Phase

ABSTRACT The role of serologic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in both the clinical and public health settings, will continue to evolve as we gain increasing insight into our immune response to the virus. Here, we evaluated four high-throughput serologic tests for detection of anti-SARS-CoV-2 IgG antibodies, from Abbott Laboratories (Abbott Park, IL), Epitope Diagnostics, Inc. (San Diego, CA), Euroimmun (Lubeck, Germany), and Ortho-Clinical Diagnostics (Rochester, NY), using a panel of serially collected serum samples (n = 224) from 56 patients with confirmed coronavirus disease 2019 (COVID-19), healthy donor sera from 2018, and a cross-reactivity serum panel collected in early 2020. The sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays in convalescent-phase serum samples collected more than 14 days post-symptom onset or post-initial positive reverse transcriptase PCR (RT-PCR) result were 92.9% (78/84), 88.1% (74/84), 97.6% (82/84), and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays were 97.3% (36/37), 73% (27/37), 94.6% (35/37), and 97.3% (36/37), respectively. Overall assay specificity/positive predictive values based on a 5% prevalence rate were 99.6%/92.8%, 99.6%/90.6%, 98.0%/71.2%, and 99.6%/92.5%, respectively, for the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays. In conclusion, we show high sensitivity in convalescent-phase sera and high specificity for the Abbott, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays. With the unprecedented influx of commercially available serologic tests for detection of antibodies against SARS-CoV-2, it remains imperative that laboratories thoroughly evaluate such assays for accuracy prior to implementation.

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