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TwinStrand Biosciences
  • Technology
  • Applications
    • AML MRD
    • Mutagenesis
    • Cellular Immunotherapy
    • Collaborate
  • Resources
    • Publications
    • Posters
    • Safety
    • Patents
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    • Our Team
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Publications

 

Featured Publications

Genetic Toxicity Testing Using Human In Vitro Organotypic Airway Cultures

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Environmental and Molecular Mutagenesis. 2021.

Genetic Toxicity Testing Using Human In Vitro Organotypic Airway Cultures

DOI: 10.1002/em.22444PubMed: 34050964

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.

AUTHORSYiying Wang, Roberta A. Mittelstaedt, Rebecca Wynne, Ying Chen, Xuefei Cao, Levan Muskhelishvili, Kelly Davis, Timothy W. Robison, Wei Sun, Elizabeth K. Schmidt,Thomas H. Smith, Zachary K. Norgaard, Charles C. Valentine, Jeffry Yaplee, Lindsey N. Williams, Jesse J. Salk, Robert H. Heflich

Enhancing the Accuracy of Next-Generation Sequencing for Detecting Rare and Subclonal Mutations

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Nature Review Genetics. 2018

Enhancing the Accuracy of Next-Generation Sequencing for Detecting Rare and Subclonal Mutations

10.1038/nrg.2017.117PubMed: 29576615

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ∼1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage.  We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

AUTHORSJesse J. Salk, Michael W. Schmitt, and Lawrence A. Loeb

Next-Generation Genotoxicology: Using Modern Sequencing Technologies to Assess Somatic Mutagenesis and Cancer Risk

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Environmental and Molecular Mutagenesis. 2019

Next-Generation Genotoxicology: Using Modern Sequencing Technologies to Assess Somatic Mutagenesis and Cancer Risk

10.1002/em.22342PubMed: 31595553

Mutations have a profound effect on human health, particularly through an increased risk of carcinogenesis and genetic disease. The strong correlation between mutagenesis and carcinogenesis has been a driving force behind genotoxicity research for more than 50 years. The stochastic and infrequent nature of mutagenesis makes it challenging to observe and to study. Indeed, decades have been spent developing increasingly sophisticated assays and methods to study these low-frequency genetic errors, in hopes of better predicting which chemicals may be carcinogens, understanding their mode of action, and informing guidelines to prevent undue human exposure. While effective, widely used genetic selection-based technologies have a number of limitations that have hampered major advancements in the field of genotoxicity. Emerging new tools, in the form of enhanced next-generation sequencing platforms and methods, are changing this paradigm. In this review, we discuss rapidly evolving sequencing tools and technologies, such as error-corrected sequencing and single cell analysis, which we anticipate will fundamentally reshape the field. In addition, we consider a variety emerging applications for these new technologies, including the detection of DNA adducts, inference of mutational processes based on genomic site and local sequence contexts, and evaluation of genome engineering fidelity, as well as other cutting-edge challenges for the next 50 years of environmental and molecular mutagenesis research.

AUTHORSJesse J. Salk and Scott R. Kennedy

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue Over a Century of Human Lifespan

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Cell Reports. 2019

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue Over a Century of Human Lifespan

doi.org/10.1016PubMed: 31269435

High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors.We show that low-frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging, and great care must be taken to distinguish tumor-derived from age associated mutations in high-sensitivity clinical cancer diagnostics.

AUTHORSJesse J. Salk, Kaitlyn Loubet-Senear, Elisabeth Maritschnegg, Charles C. Valentine, Lindsey N. Williams, Jacob E. Higgins, Reinhard Horvat, Adriaan Vanderstichele, Daniela Nachmanson, Kathryn T. Baker, Mary J. Emond, Emily Loter, Maria Tretiakova, Thierry Soussi, Lawrence A. Loeb, Robert Zeillinger, Paul Speiser, and Rosa Ana Risques
Environmental and Molecular Mutagenesis. 2021.
Yiying Wang, Roberta A. Mittelstaedt, Rebecca Wynne, Ying Chen et al.

Genetic Toxicity Testing Using Human In Vitro Organotypic Airway Cultures

DOI: 10.1002/em.22444PubMed: 34050964
PNAS. 2020
Charles C. Valentine III, Robert R. Young, Mark R. Fielden et al.

Direct Quantification of In Vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

pnas.org/content/117/52/33414
Blood Cancer Journal. 2020
Nicholas J. Short and Hagop Kantarjian et al.

Ultra-accurate Duplex Sequencing for the assessment of pretreatment ABL1 kinase domain mutations in Ph+ ALL

DOI: 10.1038/s41408-020-0329-yPubMed: 32457305
PNAS. 2019
Lawrence A. Loeb and Brendan F. Kohrn et al.

Extensive Subclonal Mutational Diversity in Human Colorectal Cancer and its Significance

DOI: 10.1016/j.ygyno.2019.11.124PubMed: 31806761
PNAS. 2019
Jiang-Cheng Shen and Ashwini S. Kamath-Loeb et al.

A High-Resolution Landscape of Mutations in the BCL6 Super-Enhancer in Normal Human B Cells

DOI: 10.1073/pnas.1914163116PubMed: 31748270
Gynecologic Oncology. 2019
Jeffrey D. Krimmel-Morrison and Talayeh S. Ghezelayagh et al.

Characterization of TP53 Mutations in Pap Test DNA of Women with and without Serous Ovarian Carcinoma

DOI: 10.1016/j.ygyno.2019.11.124PubMed: 31839337
Environmental and Molecular Mutagenesis. 2019
Jesse J. Salk and Scott R. Kennedy

Next-Generation Genotoxicology: Using Modern Sequencing Technologies to Assess Somatic Mutagenesis and Cancer Risk

10.1002/em.22342PubMed: 31595553
Nature Communication. 2019
Simonetta Andreazza and Colby L. Samstag et al.

Mitochondrially-Targeted APOBEC1 is a Potent mtDNA Mutator Affecting Mitochondrial Function and Organismal Fitness in Drosophila

DOI: 10.1038/s41467-019-10857-yPubMed: 31337756
Cell Reports. 2019
Jesse J. Salk andKaitlyn Loubet-Senear et al.

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue Over a Century of Human Lifespan

doi.org/10.1016PubMed: 31269435
eLife. 2019
Jianing Xu and Ed Reznik et al.

Abnormal Oxidative Metabolism in a Quiet Genomic Background Underlies Clear Cell Papillary Renal Cell Carcinoma

DOI: 10.7554/eLife.38986PubMed: 30924768
AACR Journals. 2019
Kathryn T Baker, Daniela Nachmanso , Shilpa Kumar, et al.

Mitochondrial DNA Mutations are Associated with Ulcerative Colitis Preneoplasia but Tend to be Negatively Selected in Cancer

DOI: 10.1158/1541-7786.MCR-18-0520PubMed: 30446624
Nature. 2018
Gannie Tzoneva and Chelsea L. Dieck et al.

Clonal Evolution Mechanisms in NT5C2 Mutant-Relapsed Acute Lymphoblastic Leukaemia

DOI: 10.1038/nature25186PubMed: 29342136
PLoS Genetics. 2018
Colby L. Samstag and Jake G. Hoekstra et al.

Deleterious Mitochondrial DNA Point Mutations are Overrepresented in Drosophila Expressing a Proofreading-Defective DNA Polymerase γ

DOI: 10.1371/journal.pgen.1007805PubMed: 30452458
Genome Research. 2018
Daniela Nachmanson and Shenyi Lian et al.

Targeted Genome Fragmentation with CRISPR/Cas9 Enables Fast and Efficient Enrichment of Small Genomic Regions and Ultra-Accurate Sequencing with Low DNA Input (CRISPR-DS)

DOI: 10.1101/gr.235291.118PubMed: 30232196
Clinical Cancer Research. 2018
Michael W. Schmitt and Justin R. Pritchard et al.

Single-Molecule Sequencing Reveals Patterns of Preexisting Drug Resistance That Suggest Treatment Strategies in Philadelphia-Positive Leukemias

DOI: 10.1158/1078-0432.CCR-18-0167PubMed: 30042204
Nature Review Genetics. 2018
Jesse J. Salk, Michael W. Schmitt, and Lawrence A. Loeb

Enhancing the Accuracy of Next-Generation Sequencing for Detecting Rare and Subclonal Mutations

10.1038/nrg.2017.117PubMed: 29576615
PNAS. 2017
Supawadee Chawanthayatham and Charles C. Valentine III et al.

Mutational Spectra of Aflatoxin B1 in vivo Establish Biomarkers of Exposure for Human Hepatocellular Carcinoma

DOI: 10.1073/pnas.1700759114PubMed: 28351974
PNAS. 2016
Kate S. Reid-Bayliss and Sarah T. Arron et al.

Why Cockayne Syndrome Patients Do Not Get Cancer Despite Their DNA Repair Deficiency

DOI: 10.1073/pnas.1610020113PubMed: 27543334
Anneals of Neurology. 2016
Jake G. Hoekstra, PhD and Michael J. Hipp et al.

Mitochondrial DNA Mutations Increase in Early Stage Alzheimer Disease and Are Inconsistent with Oxidative Damage

DOI: 10.1002/ana.24709PubMed: 27315116
eLife. 2016
Maulik R. Patel and Ganesh K. Miriyala et al.

A Mitochondrial DNA Hypomorph of Cytochrome Oxidase Specifically Impairs Male Fertility in Drosophila Melanogaster

DOI: 10.7554/eLife.16923PubMed: 27481326
PNAS. 2016
Jeffrey D. Krimmel and Michael W. Schmitt et al.

Ultra-Deep Sequencing Detects Ovarian Cancer Cells in Peritoneal Fluid and Reveals Somatic TP53 Mutations in Noncancerous Tissues

DOI: 10.1073/pnas.1601311113PubMed: 27152024
Nature Review Clinical Oncology. 2015
Michael W. Schmitt and Lawrence A. Loeb et al.

The Influence of Subclonal Resistance Mutations on Targeted Cancer Therapy

DOI: 10.1038/nrclinonc.2015.175PubMed: 26483300
PLoS One. 2015
Eun Hyun Ahn and Kensen Hirohata et al.

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing

DOI: 10.1371/journal.pone.0136216PubMed: 26305705
Neuron. 2015
Alicia M. Pickrell and Chiu-Hui Huang et al.

Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons Following Mitochondrial DNA Mutagenic Stress

DOI: 10.1016/j.neuron.2015.06.034PubMed: 26182419
Nature Methods. 2015
Michael W. Schmitt and Edward J. Fox et al.

Sequencing Small Genomic Targets with High Efficiency

DOI: 10.1038/nmeth.3351PubMed: 25849638
Nature Protocols. 2014
Scott R. Kennedy and Michael W. Schmitt et al.

Detecting Ultralow-Frequency Mutations by Duplex Sequencing

DOI: 10.1038/nprot.2014.170PubMed: 25299156
PLoS Genetics. 2013
Scott R. Kennedy and Jesse J. Salk et al.

Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage

DOI: 10.1371/journal.pgen.1003794PubMed: 24086148
PNAS. 2012
Michael W. Schmitt and Scott R. Kennedy et al.

Detection of Ultra-Rare Mutations by Next-Generation Sequencing

PNAS.1208715109PubMed: 22853953

 

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American Society of Hematology (ASH)

December 5-8, 2020 – Virtual

 

Poster Presentation

 

“Duplex Sequencing with Patient-Specific Hybrid Capture Panels Reveals Ultra-Low Frequency Measurable Residual Disease in Pediatric Acute Myeloid Leukemia”

 

Presented by Jake Higgins, Senior Scientist – December 6, 2020 7:00 a.m.-3:30 p.m. PST

 

Conference Website

 

Society of Toxicology (SOT)

March 10-14, 2019 – Baltimore, MD

 

Poster Presentations

 

 

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Conference Website

 

AACR Annual Meeting – San Diego, CA

March 29-April 3, 2019

 

Poster Presentations

 

 

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Presentations by CEO Jesse Salk, MD, PhD – AGBT 2020

Ultra-Sensitive Residual Leukemia Detection with Patient-Specific Dupex Sequencing