Dovetail Genomics, LLC

Paul Hartley

Nicholas H Putnam

Marco Blanchette

Richard E Green

Christopher John Troll

Brandon J Rice

Daniel S Rokhsar

Jonathan Stites

Martin P Powers

Andrew Fields

Ei Ei Min

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

Various approaches for generating long-distance contiguity information to facilitate contig assembly and phase determination are disclosed. Nucleic acids are assembled into complexes using binding moieties such that, when the nucleic acid backbones are cleaved, the ensuing fragments remain bound. Exposed ends are tagged and ligated either to one another or to tagging moieties such as oligo labels. Ligated junctions are sequenced, and the sequence information is used to assemble contigs into common scaffolds or to assign phase information. Various approaches to tagging the exposed ends are presented.

Tagging nucleic acids for sequence assembly

The disclosure provides methods to isolate genome or chromosome level structural information from preserved samples. In some cases, samples preserved under conditions where long-range nucleic acid information is believed to be irreparably lost, such as FFPE samples, are treated to recover nucleic acid-protein complexes stabilized as part of the sample preservation process. The complexes are processed so as to recover information regarding which nucleic acids are bound to a common complex, and the information is used to recover genomic structural information.

Recovering long-range linkage information from preserved samples

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.

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San Jose, CA, United States of America

Paul Hartley