Rubicon Genomics, Inc.

Takara Bio USA, Inc.

Emmanuel Kamberov

Vladimir L Makarov

Brendan J Tarrier

Eric Bruening

Irina Sleptsova

Takao Kurihara

Tong Sun

Jonathon H Pinter

Tim Tesmer

John P Langmore

Joseph M'Mwirichia

Marta Gonzalez-Hernandez

Amanda McNulty

Karl Hecker

Marta Gonzalez-Plasky

Jonathan H Pinter

Konstantinos Charizanis

Eric E Bruening

Joseph M M'mwirichia

John Langmore

John Pinter

Joseph M M'Mwirichia

Jon Pinter

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Compositions including a double stranded nucleic acid molecule and a stem-loop oligonucleotide

Methods of barcoding nucleic acids, such as genomic DNA, are provided herein. In some embodiments, a fragment of genomic DNA may comprise a first and a second barcode.

Barcoding nucleic acids

Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3'-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.

Coupling adaptors to a target nucleic acid

Provided herein are compositions for and methods of generating ligation-competent nucleic acids. In some aspects, the compositions comprise Exonuclease III, T4 DNA Polymerase, Klenow, and/or T4 polynucleotide kinase.

Methods and compositions for repair of DNA ends by multiple enzymatic activities

The present invention is directed to methods to prepare a DNA molecule or a plurality of DNA molecules by random fragmentation. In some embodiments, the present invention regards preparing a template for DNA sequencing by random fragmentation. In specific embodiments, the random fragmentation comprises chemical fragmentation, mechanical fragmentation, or enzymatic fragmentation. In further specific embodiments, a universal sequence is attached to the 3' end of the DNA fragments, such as by ligation of an adaptor sequence or by homopolymeric tailing with terminal deoxynucleotidyltransferase. In other embodiments, a library is prepared with methods of the present invention.

DNA amplification and sequencing using DNA molecules generated by random fragmentation

Kits including stem-loop oligonucleotides for use in preparing nucleic acid molecules

The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5' constant sequence and a 3&#x2032; variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3&#x2032; hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules.

Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation

The present invention is directed generally to the use of stem-loop oligonucleotides comprising an inverted repeat, a loop, and at least one degradable site or site capable of becoming a degradable site in the preparation of DNA molecules, such as a library. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

Methods for processing and amplifying nucleic acids

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Ann Arbor, MI, United States of America

Emmanuel Kamberov