Leland Stanford Junior University

Arc Bio, LLC

Carlos Daniel Bustamante

Meredith L Carpenter

Stephane B Gourguechon

William J Greenleaf

Jason D Buenrostro

Michael P Snyder

Euan A Ashley

Frederick E Dewey

Matthew Thomas Wheeler

Fouad Zakharia

Suyash Shringarpure

Provided herein are methods and compositions for depleting targeted nucleic acid sequences from a sample, enriching for sequences of interest from a sample, and/or partitioning of sequences from a sample. The methods and compositions are applicable to biological, clinical, forensic, and environmental samples.

Compositions and methods for targeted depletion, enrichment, and partitioning of nucleic acids using CRISPR/Cas system proteins

Provided herein is a method for capturing DNA molecules in solution. The method may comprise: extracting DNA from a sample that comprises endogenous DNA and environmental DNA to produce extracted DNA; ligating universal adaptors to the extracted DNA; hybridizing the extracted DNA, in solution, with affinity-tagged RNA probes generated by: in vitro transcribing a library of fragmented reference genomic DNA that has been ligated to an RNA promoter adaptor, in the presence of an affinity-tagged ribonucleotide; binding the product with a capture agent that is tethered to a substrate in the presence of RNA oligonucleotides that are complementary to the adaptors, thereby capturing the hybridized DNA molecules on the substrate; washing the substrate to remove any unbound DNA molecules; and releasing the captured DNA molecules. A kit for performing the method is also provided.

Enrichment of DNA sequencing libraries from samples containing small amounts of target DNA

Described herein is a method for isolating microbial DNA from a sample that comprises host DNA and microbial DNA. In some embodiments, the method may comprise: obtaining a tagged DNA sample, wherein the tagged DNA sample contains host DNA and microbial DNA, both comprising an appended universal adaptor; b) hybridizing the extracted DNA, in solution, with affinity-tagged RNA probes generated by in vitro transcribing, in the presence of an affinity-tagged ribonucleotide, a library of fragmented host DNA that has been ligated to an RNA promoter adaptor; c) binding the product of step b) with a capture agent that is tethered to a substrate, in the presence of RNA oligonucleotides that are complementary to or have the same sequence as one or more strands of the universal adaptor, thereby capturing the host DNA on the substrate; and d) collecting the unbound DNA, wherein the unbound DNA comprises the microbial DNA.

Partitioning of DNA sequencing libraries into host and microbial components

A novel phasing algorithm harnesses sequencing read information from next generation sequencing technologies to guide and improve local haplotype reconstruction from genotypes. Techniques include determining correlated occurrences of single nucleotide polymorphisms (SNPs) in genes of a population of individuals. A plurality of sequences of nucleotide bases in one or more individuals from the populations of individuals is determined based on ultra-high throughput sequencing of a sample from the one or more individuals. Haplotypes included in the population of individuals are determined based on both the correlated occurrences and the plurality of sequences. The inclusion of paired end read data is especially advantageous for the phasing of rare variants, including singletons.

Techniques for determining haplotype by population genotype and sequence data

Techniques to determine if an individual is a member of an ensemble contributing to combined genomic data include determining a site frequency spectrum (SFS) for alleles in the combined genomic data, and a number N of individuals contributing to the combined genomic data. Based on the SFS and N, both a number of queries NQ and a number of yeses NY are determined. NY is a number of yes responses to NQ queries, which indicates a particular likelihood that the individual is a member of the ensemble. Output is based on at least one of NQ and NY. For example, fewer than NQ queries from a single source are answered from a beacon. Alternatively, the likelihood that an individual is a member of the ensemble is determined based on responses to NQ queries about NQ different alleles present in the individual and NY.

Techniques for determining whether an individual is included in ensemble genomic data

In an embodiment of the present invention, three novel human reference genome sequences were developed based on the most common population-specific DNA sequence ('major allele'). Methods were developed for their integration into interpretation pipelines for highthroughput whole genome sequencing.

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Emerald Hills, CA, United States of America

Carlos Daniel Bustamante