Agilent Technologies, Inc.

Leland Stanford Junior University

Brian Jon Peter

Robert A Ach

Nicholas Michael Sampas

Joel Myerson

Bo U Curry

John Mannion

Holly Hogrefe

Gusti Zeiner

Jeffrey Robert Sampson

Jayati Ghosh

Zoltan Timar

Dan-Hui Dorothy Yang

Bahram Arezi

Derek Lee Lindstrom

David Taussig

Anya Tsalenko

Alicia Scheffer-Wong

Samuel So

Michael Walter

Hongbo Sun

Bram Herman

Carolina Caffaro

Arjun Vadapalli

Mei-Sze Chua

Described herein is an adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary. A method, as well as a kit for practicing the method, are also provided.

Use of transposase and Y adapters to fragment and tag DNA

A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

Nucleic acid enrichment using Cas9

A method for making an asymmetrically-tagged sequencing library is provided. In some embodiments, the method may comprise: obtaining a symmetrically-tagged library of cDNA or genomic DNA fragments, hybridizing a tailed first primer to the 3' sequence tag of the library and extending the same to produce primer extension products, and amplifying the primer extension products using a pair of tailed primers to produce asymmetrically-tagged library.

Method for making an asymmetrically-tagged sequencing library

In some embodiments, the amplification method may comprise producing a reaction mix comprising: a nucleic acid sample, a polymerase, nucleotides, a forward primer that hybridizes to a sequence in the bottom strand of a fragment in the sample, and a reverse primer. The reverse primer has a hairpin structure comprising a loop, a stem and a 3' overhang of at least 8 nucleotides, wherein the 3&#x2032; overhang hybridizes to a sequence in the top strand of the fragment. Subjecting the reaction mix at least two rounds of denaturation, renaturation and primer extension conditions results in extension the forward and reverse primers to produce an amplification product that contains: a double stranded region comprising a nick adjacent to the 5&#x2032; end of the reverse primer, and the loop of the first hairpin primer. Primer sets and kits for performing the methods are also provided.

Hairpin-mediated amplification method

A method for sequencing a nucleic acid is provided. In certain embodiments the method comprises obtaining a duplex comprising a nucleic acid and a primer, wherein the primer has a nuclease resistant 3' end, combining the duplex with a chain terminator nucleotide and a proof-reading polymerase to produce a reaction in which the polymerase idles on the added chain terminator nucleotide, identifying the chain terminator nucleotide added to the end of the primer; and adding a nuclease-resistant nucleotide to the end of the primer after the polymerase has idled on and removed the added chain terminator nucleotide, thereby producing a duplex comprising the template and an extended primer that has a nuclease resistant 3&#x2032; end.

Polymerase idling method for single molecule DNA sequencing

A method for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

Method for fragmenting genomic DNA using CAS9

This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided.

Target enrichment and labeling for multi-kilobase DNA

Provided herein is a method for enriching a target nucleic acid molecule. In one embodiment, the method may involve hybridizing a C-probe to a strand of a target nucleic acid to produce a complex, enzymatically removing any 3' overhanging end from the target nucleic acid of the complex to produce a 3&#x2032; hydroxyl group at the 3&#x2032; end; extending the 3&#x2032; end of the first sequence using the oligonucleotide sequence of the C-probe as a template; enzymatically removing any 5&#x2032; overhanging end from the target nucleic acid, either before or after the extending step, to produce an 5&#x2032; phosphate group at the end of the second sequence; and ligating the 5&#x2032; phosphate group at the end of the second sequence to the 3&#x2032; hydroxyl group at the end of the first sequence to produce a circular DNA molecule that contains the target sequence and the complement of the oligonucleotide sequence.

C-probe libraries for DNA target enrichment

This disclosure provides, among other things, a method for analyzing a planar cellular sample. In some embodiments, the method comprises: (a) indirectly or directly attaching nucleic acid tags to binding sites in a planar cellular sample; (b) contacting the planar cellular sample with a solid support comprising an array of spatially addressed features that comprise oligonucleotides, wherein each oligonucleotide comprises a molecular barcode that identifies the feature in which the oligonucleotides is present; (c) hybridizing the nucleic acid tags, or a copy of the same, with the oligonucleotides to produce duplexes; and (d) extending the oligonucleotides in the duplexes to produce extension products that each comprises (i) a molecular barcode and (ii) a copy of a nucleic acid tag. Other embodiments, e.g., kits and the like, are also described.

Spatial molecular barcoding of in situ nucleic acids

This disclosure provides, among other things, a nanofluidic device sensing device is provided. In certain embodiments, the device contains: a) a channel comprising a floor and a ceiling, b) an array of charge sensors in the floor and/or ceiling of the channel, arranged along the longitudinal axis of the channel; c) a capture area in the floor and/or ceiling of the channel at the entrance end of the channel; and d) a first electrode and a second electrode, wherein the first and second electrodes are positioned to provide an electrophoretic force along the longitudinal axis of the channel. Other embodiments, e.g., methods, are also described.

Nanofluidic device for charge analysis of straightened molecules

A method for analyzing a sequence comprising a SNP site is provide. In general terms, the method comprises: a) contacting a first DNA sample with a first restriction enzyme to provide DNA fragments, wherein: i) the first restriction enzyme cleaves the sequence only if a first allele of a SNP is present at the SNP site; b) end-labeling the DNA fragments to produce an end-labeled sample; c) hybridizing the end-labeled sample to an array comprising a probe sequence; and d) comparing the amount of hybridization between the digested sample and the probe sequence to a reference signal.

Analysis of single nucleotide polymorphisms using end labeling

A method for determining the ploidy of a test genome is provided. In some embodiments, the method may comprises: a) obtaining a plurality of ratios for polymorphisms that are distributed throughout a test genome, wherein each of the ratios is a ratio of the measured copy number of uncut allele in a polymorphic site relative to the measured copy number of the uncut allele in the reference sample; b) taking the log of the ratios and plotting a distribution of the reference corrected log ratios of the SNP probes; and c) determining the ploidy of said the genome based on the number of peaks in that distribution.

Method for determining ploidy of a cell

Certain embodiments described in this disclosure relate to a method of sample analysis. In certain cases, the method comprises: a) contacting a genomic sample comprising double-stranded genomic DNA with a first restriction endonuclease that recognizes a nucleotide sequence that comprises a SNP site in the double stranded genomic DNA, wherein: i. the restriction endonuclease cleaves the genomic DNA at the sequence regardless of the allele of the SNP present at the SNP site; and ii. cleavage of the sequence by the restriction enzyme creates a 5' overhang that comprises the SNP site; b) contacting the digested genomic sample with a extension enzyme and a first labeled nucleotide that is used by the extension enzyme to fill in the overhang only if the overhang comprises a first allele of the SNP.

Growing community of inventors

Los Altos, CA, United States of America

Brian Jon Peter