University of California

Intel Corporation

Dovetail Genomics, LLC

Other

Cleanalert, LLC

Ford Global Technolgoies, LLC

Obsidian Products, LLC

Richard E Green

Liana F Lareau

Mark T Bohr

Stephen T Chambers

Shahriar S Ahmed

Daniel S Rokhsar

Anand S Murthy

Paul Hartley

Ebrahim Andideh

Lawrence N Brigham

Marco Blanchette

William Sherman, Iii

Steven E Brenner

James Adam Drozdowski

Andrew Arthur Brown

Joseph Lee Buchwitz

Norton Herrick

Christopher John Troll

Christopher Vollmers

Lacey Bruschke

Joshua D Kapp

Jacob Peter Stencel

Erin Wathen

R Tyler Hillman

Benjamin P Lewis

Ei Ei Min

Balaji Sundararaman

Bruce G Green

Samuel H Vohr

Roger Volden

Mason Otto Suksumake

Theron Palmer

Samuel Vohr

David Saloff

The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure further provides methods for haplotype phasing and meta-genomics assemblies.

Methods for genome assembly and haplotype phasing

Disclosed herein are methods, compositions and systems that facilitate accurate phasing of sequence data such as genomic sequence data through the segmentation and rearrangement of nucleic acid molecules in such a way as to preserve individual molecules' phase or physical linkage information. This is variously accomplished by binding molecules independent of their phosphodiester backbones, cleaving the molecules, ligating, and sequencing the molecules through long-read sequencing technology to recover segment sequence information spanning at least more than one segment.

Generation of phased read-sets for genome assembly and haplotype phasing

Provided are methods of nucleic acid sequencing. The methods include producing a circularized DNA including a full-length cDNA and a known heterologous sequence, and performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence. The methods further include obtaining a raw sequencing read of the concatemer using a nanopore, identifying the repeating segments in the raw sequencing read, and producing a consensus sequence of the full-length cDNA based on the sequences of the repeating segments. Computer-readable media, computing devices, and systems that find use, e.g., in practicing the methods of the present disclosure are also provided.

Nucleic acid sequencing methods and computer-readable media for practicing same

The disclosure provides for methods to form compact cross-linked polynucleotide/protein structures that can then be labeled using a barcoded oligonucleotide array in order to reconstruct physical linkage and/or genomic proximity (and phase) of polynucleotide fragments.

Methods for labeling DNA fragments to reconstruct physical linkage and phase

Provided are methods of producing nucleic acid libraries. The methods include combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA), an adapter oligonucleotide, and a splint oligonucleotide, to form complexes including the splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA and to the adapter oligonucleotide. An end of the first adapter oligonucleotide is adjacent to an end of the first terminal region of the SSB-bound ssNA, and the methods may further include covalently linking the adjacent ends. Also provided are compositions and kits that find use, e.g., in practicing the methods of the present disclosure.

Methods of producing nucleic acid libraries and compositions and kits for practicing same

Aspects of the present disclosure include methods of making barcoded solid supports. In some embodiments, the methods include producing a concatemer by rolling circle amplification (RCA) of a circular nucleic acid template, where the circular nucleic acid template includes a barcode and a stem-loop forming region, and where the concatemer includes a plurality of linked units, each unit including the barcode and a stem-loop structure formed from the stem-loop forming region. Such methods further include disposing the concatemer on a solid support to produce a barcoded solid support including a plurality of the stem-loop structures extending from the surface of the solid support. The methods may further include treating the stem-loop structures with an agent that produces stem structures having ends compatible with target nucleic acids, and attaching the target nucleic acids to the stem structures. Barcoded solid supports and methods of using the barcoded solid supports are also provided.

Barcoded solid supports and methods of making and using same

Disclosed are methods for testing biological samples containing genomic nucleic acids obtained from an organism having a genome, such as a human genome. It is often desirable to analyze a DNA sample or more than one, different DNA samples, to determine whether the sample comes from one individual or two individuals. The present method requires very low amounts of DNA and can use partial sequences of DNA fragments. Partial sequences are analyzed for the presence of polymorphisms (e.g. SNP's) that can be mapped to a reference SNP map. The distance between similar SNPS, which are genetically linked, can be used to statistically determine a likelihood of identity of individuality in a sample.

Method for determining relatedness of genomic samples using partial sequence information

Provided are methods of producing nucleic acid libraries. In certain aspects, the methods include combining target nucleic acids (e.g., 5' phosphorylated nucleic acids) and an oligonucleotide pool. Oligonucleotides of the oligonucleotide pool may include complementarity regions of varying length and nucleotide sequence, and a complementarity region identification sequence. In such aspects, the combining is under conditions in which oligonucleotides of the oligonucleotide pool hybridize to nucleic acids of the target nucleic acids (e.g., 5&#x2032; phosphorylated nucleic acids) having overhang regions that are complementary in sequence and have corresponding lengths with respect to the complementarity regions of the oligonucleotides. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

Methods of producing nucleic acid libraries

Methods for labeling DNAa fragments to reconstruct physical linkage and phase

Disclosed herein are methods, compositions and systems that facilitate accurate phasing of sequence data such as genomic sequence data through the segmentation and rearrangement of nucleic acid molecules in such a way as to preserve individual molecules phase or physical linkage information. This is variously accomplished by binding molecules independent of their phosphodiester backbones, cleaving the molecules, ligating, and sequencing the molecules through long-read sequencing technology to recover segment sequence information spanning at least more than one segment.

The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure provides methods for haplotype phasing and meta-genomics assemblies. The disclosure provides a streamlined method for accomplishing these tasks, such that intermediates need not be labeled by an affinity label to facilitate binding to a solid surface. The disclosure also provides methods and compositions for the de novo generation of scaffold information, linkage information, and genome information for unknown organisms in heterogeneous metagenomic samples or samples obtained from multiple individuals. Practice of the methods can allow de novo sequencing of entire genomes of uncultured or unidentified organisms in heterogeneous samples, or the determination of linkage information for nucleic acid molecules in samples comprising nucleic acids obtained from multiple individuals.

Methods for genome assembly, haplotype phasing, and target independent nucleic acid detection

Growing community of inventors

Santa Cruz, CA, United States of America

Richard E Green