University of Washington

Brandeis University

Twinstrand Biosciences, Inc.

Jesse J Salk

Lawrence A Loeb

Michael W Schmitt

Lawrence J Wangh

John E Rice

Kenneth E Pierce

Cristina Hartshorn

J Aquiles Sanchez

Scott R Kennedy

Arthur Reis

Elizabeth Schmidt

Rosa Ana Risques

Daniela Nachmanson

Michael Hipp

Arthur Henry Reis, Jr

Charles Clinton Valentine, Iii

Aquiles J Sanchez

Kenneth Pierce

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 approximately 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, a method Duplex Consensus Sequencing (DCS) is provided. 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 will result in errors in only one strand. This method uniquely capitalizes on the redundant information stored in double-stranded DNA, thus overcoming technical limitations of prior methods utilizing data from only one of the two strands.

Methods of lowering the error rate of massively parallel DNA sequencing using duplex consensus sequencing

The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications. In some embodiments, highly accurate, error corrected and massively parallel sequencing of nucleic acid material is possible using a combination of uniquely labeled strands in a double-stranded nucleic acid complex in such a way that each strand can be informatically related to its complementary strand, but also distinguished from it following sequencing of each strand or an amplified product derived therefrom. In various embodiments, this information can be used for the purpose of error correction of the determined sequence.

Methods for targeted nucleic acid sequence enrichment with applications to error corrected nucleic acid sequencing

Methods for characterizing genome editing, clonal expansion and associated reagents for use in such methods are disclosed herein. Some embodiments of the technology are directed to characterizing a population of cells following an engineered genomic editing event, that includes in some embodiments characterizing genomic alterations occurring at both intended and unintended genomic loci within the genome of the populations of cells. Other embodiments are directed to utilizing Duplex Sequencing for assessing a clonal selection in mixed cell populations and/or cell populations following a genomic editing event. Further examples of the present technology are directed to methods for detecting and assessing clonal expansion of cells following a genomic editing event.

Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications

Methods for making and using reagents and adapters for use in nucleic acid sequencing applications are disclosed herein. In several embodiments, adapters and reagents and the methods of making such adapters and reagents can be used for Duplex Sequencing applications.

Reagents and adapters for nucleic acid sequencing and methods for making such reagents and adapters

Disclosed herein are adapter nucleic acid sequences, double-stranded complexed nucleic acids, compositions, and methods for sequencing a double-stranded target nucleic acid with applications to error correction by duplex sequencing.

Adapters, methods, and compositions for duplex sequencing

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Seattle, WA, United States of America

Jesse J Salk