Agilent Technologies, Inc.

Robert A Ach

Brian Jon Peter

Gusti Zeiner

Brian Phillip Smart

Nicholas Michael Sampas

Douglas J Dellinger

Joel Myerson

Holly Hogrefe

Mark Andrew Troll

Jeffrey Robert Sampson

Zoltan Timar

Kristin Briana Bernick

Bahram Arezi

SueAnn C Dahm

Derek Lee Lindstrom

Diane D Ilsley

Carsten Carstens

Alicia Scheffer-Wong

David Taussig

Carolina Caffaro

Bernd Buehler

Michael Walter

Bram Herman

Mistuni Ghosh

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 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

Among other things, this disclosure provides a method of detecting a target nucleic acid. Aspects of the method include: (a) obtaining a labeled nucleic acid probe that is complementary to a target nucleic acid, wherein the probe comprises a capture tag; (b) hybridizing the probe with the target nucleic in a fixed cell, in situ, to produce a duplex; (c) linking the probe in the duplex to a peroxidase conjugate via the capture tag to produce a peroxidase-labeled duplex; and (d) incubating the peroxidase-labeled duplex with a peroxidase substrate, wherein the peroxidase activity of the peroxidase conjugate catalyzes deposition of the substrate in the vicinity of the duplex, thereby producing a detectable signal.

Signal amplification of fluorescence in situ hybridization

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

Provided herein is a method of sample analysis. In certain embodiments, the method comprises: a) cross-linking protein of a cell using a first compound to produce a first cross-linked product comprising cross-linked protein, and RNA; b) contacting the first cross-linked product and a second compound under conditions by which an oligonucleotide portion of the second compound hybridizes to the RNA; c) activating a reaction the first and second compound, thereby covalently crosslinking the oligonucleotide to the cross-linked protein to produce a second cross-linked product; d) isolating the second cross-linked product using an affinity tag; and e) analyzing the isolated second cross-linked product. Compounds for performing the method are also provided.

Isolation of RNA-protein complexes using cross-linking reagents and oligonucleotides

A method of sample analysis is provided. In certain embodiments, the method may comprise: a) contacting under primer extension conditions a genomic sample comprising a test genome with a set of at least ten sequence-specific primers that are complementary to sites in only one strand of a reference chromosomal region, to produce primer extension products, and b) analyzing the primer extension products to identify a chromosomal rearrangement in the test genome.

Method for genome analysis

A method of processing an RNA sample is provided. In certain embodiments, the method may comprise: a) obtaining a fragmented RNA sample comprising: i. RNA fragments of long RNA molecules; and ii. unfragmented short RNA; and b) contacting said fragmented RNA sample with a first adaptor in the presence of a RtcB ligase, thereby producing a ligated RNA sample comprising adaptor-ligated fragments of long RNA. A kit for performing the method is also provided.

Ligation method employing RtcB

Provided herein is a method of preparing an RNA sample comprising: a) obtaining an RNA sample comprising: i. long RNA molecules that may be unfragmented or fragmented to contain 5'-OH group and a 2&#x2032;-3&#x2032;-cyclic phosphate group; and ii. short RNA molecules that comprise a 5&#x2032; phosphate group and a 3&#x2032; OH group; and b) contacting the RNA sample with an adaptor comprising either a 2&#x2032;-PO group and 3&#x2032;-OH group or a 2&#x2032;,3&#x2032;-cyclic phosphate group in the presence of a eukaryotic tRNA ligase, thereby producing a ligated RNA sample in which a) the short RNA molecules are selectively ligated to the adaptor or b) the short RNA molecules and long RNA fragments are selectively ligated to the adaptor.

Ligation method employing eukaryotic tRNA ligase

Provided herein is method comprising: contacting an initial RNA sample containing a population of different RNA molecules with a divalent cation and a set of DNAzymes that are designed to cleave multiple target RNAs in the initial sample, thereby producing a product RNA sample that comprises: a) uncleaved RNA molecules and b) cleaved RNA fragments that contain a 2',3&#x2032;-cyclic-phosphate and a 5&#x2032; hydroxyl as the result of DNAzyme cleavage.

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San Francisco, CA, United States of America

Robert A Ach