Leland Stanford Junior University

Guardant Health, Inc.

Cz Biohub Sf, Llc.

William J Greenleaf

Jason D Buenrostro

Howard Y Chang

Paul Giresi

Andrew Kennedy

Curtis J Layton

Carlos Daniel Bustamante

Meredith L Carpenter

Ariel Jaimovich

Stefanie Ann Ward Mortimer

Oscar Westesson

Yupeng He

Kara Brower

Sandy Klemm

Steven M Block

Matthew Schultz

Xingqi Chen

Polly Fordyce

A novel method for displaying proteins and peptides is disclosed in which individual proteins or peptides remain associated with the DNA encoding them. Proteins or peptides can be generated by in vitro translation of DNA templates, either free in solution or arrayed on a solid support, such that the proteins or peptides remain immobilized on their DNA templates. In particular, high throughput sequencing can be combined with high throughput functional characterization of encoded proteins and peptides, wherein the identity of each protein or peptide is determined by DNA sequencing, and functional studies are carried out directly on each protein or peptide while immobilized on the DNA template encoding it. The methods of the invention should find numerous applications, for example, in high throughput genetic or pharmacological screening, epitope mapping, and protein engineering and directed evolution.

Devices and methods for display of encoded peptides, polypeptides, and proteins on DNA

The present disclosure provides compositions and methods related to analyzing DNA, such as cell-free DNA. In some embodiments, the cell-free DNA is from a subject having or suspected of having cancer and/or the cell-free DNA includes DNA from cancer cells. In some embodiments, the DNA is partitioned into a first subsample and a second subsample, wherein the first subsample comprises DNA with a nucleotide modification (e.g., a cytosine modification) in a greater proportion than the second subsample, and the second subsample is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample, and the DNA is sequenced in a manner that distinguishes the first nucleobase from the second nucleobase in the DNA of the second subsample.

Compositions and methods for analyzing DNA using partitioning and base conversion

The invention provides methods of performing multi-parameter analysis of single cells. Specifically, the present invention is based, in part, on leveraging a separation of reaction volume scales to conduct high-throughput single cell multi-parameter measurements and library preparation on the same single cell using double emulsion micro-droplets and sorting using flow cytometry.

Method to perform high-throughput single cell genomic and phenotypic analyses

In an aspect, the present disclosure provides a method for determining a methylation status comprises: providing a biological sample of nucleic acid molecules; partitioning at least a subset of the nucleic acid molecules in the biological sample based on the methylation status of the nucleic acid molecules into a plurality of partitioned sets; digesting at least a subset of the one or more partitioned sets in the plurality of partitioned sets with at least one methylation sensitive restriction enzyme; enriching at least a subset of the nucleic acid molecules in the plurality of partitioned sets for genomic regions of interest, wherein the at least a subset of the nucleic acid molecules comprises digested nucleic acid molecules in the one or more partitioned sets; and determining methylation status at one or more genetic loci of the nucleic acid molecules in at least one of the partitioned sets.

Methods and systems to improve the signal to noise ratio of DNA methylation partitioning assays

In an aspect, a method for detecting the presence or absence of cancer in a subject comprises: (a) obtaining samples from the subject from different time points, wherein a first sample obtained at first time point is a polynucleotide sample from a tumor tissue specimen and a second sample obtained at second time point is a cell-free polynucleotide sample from a blood sample; (b) processing polynucleotides from each of the samples, comprising: i) tagging at least a portion of the polynucleotides, thereby generating tagged parent polynucleotides; ii) amplifying at least a portion of the tagged parent polynucleotides to generate progeny polynucleotides; iii) enriching at least a portion of the progeny polynucleotides for target genomic regions; thereby generating enriched polynucleotides; and iv) sequencing at least a portion of the enriched polynucleotides to generate sequencing reads; and (c) analyzing genomic regions for at least one epigenetic feature from the sequencing reads.

Methods and systems for improving patient monitoring after surgery

Provided herein is a DNA analysis method comprising partitioning a sample into at least a first subsample and a second subsample, wherein the first subsample comprises DNA (e.g., cell-free DNA) with a cytosine modification in a greater proportion; the first subsample undergoes a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA of the first subsample; and DNA is sequenced to distinguish the first nucleobase from the second nucleobase. Also provided is a combination comprising first and second populations of captured DNA, wherein the first population comprises or was derived from DNA with a cytosine modification in a greater proportion than the second population, and wherein the first population comprises a form of a first nucleobase originally present in the DNA with altered base pairing specificity and a second nucleobase without altered base pairing specificity.

Compositions and methods for analyzing cell-free DNA in methylation partitioning assays

Methods for labeling and imaging the accessible genome using a transposase are disclosed. In some embodiments, a bifunctional transposase complex or transposome is used to insert adaptors comprising chemical tags selectively at accessible sites in the genome where active regulatory DNA is located. Various chemical tags can be used for labeling DNA at insertion sites, including, for example, fluorescent dyes for fluorescence imaging, metal particles for electron microscopy or magnetic manipulation of DNA, isotopic labels, or biotin or other ligands, haptens, substrates, or inhibitors that are recognized by streptavidin, antibodies, enzymes, or receptors. Labeling DNA in this manner can be used to provide spatial information regarding the positioning of regulatory DNA in the genome and makes possible the imaging and sorting of cells based on the status of their regulatory DNA.

Transposase-mediated imaging of the accessible genome

Disclosed herein are compositions and methods for isolating DNA, such as cell-free DNA (cfDNA). In some embodiments, the cell-free DNA is from a subject having or suspected of having cancer and/or the cell-free DNA comprises DNA produced by a tumor. In some embodiments, the DNA isolated by the method is captured using a sequence-variable target region set and an epigenetic target region set, wherein the sequence-variable target region set is captured with a greater capture yield than the epigenetic target region set. In some embodiments, captured cfDNA of the sequence-variable target region set is sequenced to a greater depth of sequencing than captured cfDNA of the epigenetic target region set.

Compositions and methods for isolating cell-free DNA

Provided herein is a method for analyzing polynucleotides such as genomic DNA. In certain embodiments, the method comprises: (a) treating chromatin isolated from a population of cells with an insertional enzyme complex to produce tagged fragments of genomic DNA; (b) sequencing a portion of the tagged fragments to produce a plurality of sequence reads; and (c) making an epigenetic map of a region of the genome of the cells by mapping information obtained from the sequence reads to the region. A kit for performing the method is also provided.

Transposition of native chromatin for personal epigenomics

A method for displaying proteins and peptides is disclosed in which individual proteins or peptides remain associated with the DNA encoding them. Proteins or peptides can be generated by in vitro translation of DNA templates, either free in solution or arrayed on a solid support, such that the proteins or peptides remain immobilized on their DNA templates. In particular, high throughput sequencing can be combined with high throughput functional characterization of encoded proteins and peptides, wherein the identity of each protein or peptide is determined by DNA sequencing, and functional studies are carried out directly on each protein or peptide while immobilized on the DNA template encoding it. The methods of the invention should find numerous applications, for example, in high throughput genetic or pharmacological screening, epitope mapping, and protein engineering and directed evolution.

Devices and methods for display of encoded peptides, PolyPeptides, and proteins on DNA

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.

Growing community of inventors

Menlo Park, CA, United States of America

William J Greenleaf