Johns Hopkins University

Duke University

Other

The University of Texas System

The State University of New York

Case Western Reserve University

Nickolas Papadopoulos

Bert Vogelstein

Kenneth W Kinzler

Luis Diaz

Hai Yan

Isaac A Kinde

Darell D Bigner

Drew M Pardoll

Suzanne L Topalian

Dung Le

Victor E Velculescu

Gregory J Riggins

Sian Jones

Ralph Hruban

Chetan Bettegowda

Rebecca J Leary

Yuxuan Wang

Xiaosong Zhang

Chien-Tsun Kuan

D Williams Parsons

Jimmy Cheng-Ho Lin

Philipp Angenendt

Giovanni Parmigiani

Rachel Karchin

Yuchen Jiao

Shibin Zhou

Craig A Rosen

Steven M Ruben

Nicholas C Nicolaides

Mark D Adams

Ying-Fei Wei

Claire M Fraser

Sanford D Markowitz

Ewen F Kirkness

William A Haseltine

Meng Li

Robert D Fleischmann

William G Nelson

Rebecca A Fuldner

Scott E Kern

Antony Rosen

Srinivasan Yegnasubramanian

Livia Angela Casciola-Rosen

Michael Goggins

Jian Wu

Nishant Agrawal

Simeon Springer

Anirban Maitra

Zachary James Reitman

Joshua Cohen

Alan Meeker

Patrick J Killela

George J Netto

Roger McLendon

Renyuan Bai

Arthur P Grollman

Verena Staedtke

Erika Darrah

Samir Hanash

Ami A Shah

Qing Wang

Karin Sundfeldt

Bjorg Kristjansdottir

James R Eshleman

Donald William Parsons

Emily Han-Chung Hsiue

Christine Joseph

Michael S Hwang

Jacqueline Douglass

Cristian Tomasetti

Christopher Douville

Anne Marie Lennon

Kathleen Dickman

Georges Jabboure Netto

Margaret Hoang

Hanno Matthaei

Donald Williams Parsons

Yiping He

Marco Dal Molin

Alison Klein

Austin Mattox

Darell Bigner

Victor Velculescu

Zachary J Reitman

Williams Parsons

Kenneth Kinzler

Jimmy Chang-Ho Lin

Provided herein are systems, kits, compositions and methods for sequencing library preparation and sequencing workflow (e.g., for the identification of mutations). In certain embodiments, provides herein systems and methods to identically barcode both strands of templates, and PCR-based enrichment of each strand that does not require hybridization capture.

Methods and materials for assessing nucleic acids

Bottleneck Sequencing System (BotSeqS) is a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately prior to library amplification. BotSeqS can be used to show age and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal tissues can vary by several orders of magnitude, depending on biologic and environmental factors. BotSeqS has been used to show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, BotSeqS has shown that the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them.

Bottleneck sequencing

We queried DNA from saliva or plasma of 93 HNSCC patients, searching for somatic mutations or human papillomavirus genes, collectively referred to as tumor DNA. When both plasma and saliva were tested, tumor DNA was detected in 96% (95% CI, 84% to 99%) of 47 patients. The fractions of patients with detectable tumor DNA in early- and late-stage disease were 100% (n=10) and 95% (n=37), respectively. Saliva is preferentially enriched for tumor DNA from the oral cavity, whereas plasma is preferentially enriched for tumor DNA from the other sites. Tumor DNA in the saliva and plasma is a valuable biomarker for detection of HNSCC.

Head and neck squamous cell carcinoma assays

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called 'Safe-SeqS' for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Safe sequencing system

Provided herein are methods and materials for detecting and/or treating subject (e.g. a human) having cancer. In some embodiments, methods and materials for identifying a subject as having cancer (e.g., a localized cancer) are provided in which the presence of member(s) of two or more classes of biomarkers are detected. In some embodiments, methods and materials for identifying a subject as having cancer (e.g. a localized cancer) are provided in which the presence of member(s) of at least one class of biomarkers and the presence of aneuploidy are detected. In some embodiments, methods described herein provide increased sensitivity and/or specificity in the detection of cancer in a subject (e.g. a human).

Methods and materials for assessing and treating cancer

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

Rapid aneuploidy detection

As cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we assessed the cerebrospinal fluid (CSF) that bathes the CNS for tumor DNA, here termed CSF-tDNA. The results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.

Detection of tumor-derived DNA in cerebrospinal fluid

This document provides methods and materials for assessing a mammal having or suspected of having cancer and/or for treating a mammal having cancer. For example, molecules including one or more antigen-binding domains (e.g., a single-chain variable fragment (scFv)) that can bind to a modified peptide (e.g., a tumor antigen), as well as method for using such molecules, are provided.

MANAbodies and methods of using

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called 'Safe-SeqS' for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant ('super-mutants') if &#x2265;95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Blockade of immune checkpoints such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) shows promise in patients with cancer. Inhibitory antibodies directed at these receptors have been shown to break immune tolerance and promote anti-tumor immunity. These agents work particularly well in patients with a certain category of tumor. Such tumors may be particularly susceptible to treatment because of the multitude of neoantigens which they produce.

Checkpoint blockade and microsatellite instability

TERT promoter mutations occur in both papillary and flat lesion bladder cancers, are the most frequent genetic alterations identified to date in noninvasive precursor lesions of the bladder, are detectable in urine, and appear to be strongly associated with bladder cancer recurrence. The TERT promoter mutations are useful urinary biomarker for both the early detection and monitoring of bladder neoplasia.

TERT promoter mutations in urothelial neoplasia

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Towson, MD, United States of America

Nickolas Papadopoulos