Leland Stanford Junior University

Hanlee P Ji

Ronald W Davis

Yang Liu

Billy Tsz Cheong Lau

Jason D Buenrostro

Giwon Shin

Samuel Myllykangas

Robert W Dutton

Kee-Hyun Paik

Laura Miotke

HoJoon Lee

Anna McGinty Cushing

Jason Buenrostro

Billy Tsz Lau

A method for determining if a tumor has a mutation in a microsatellite is provided. In some embodiments, the method may comprise: (a) isolating genomic DNA from a tumor sample and a non-tumor sample from the same patient to produce: i. a sample of tumor DNA and ii. a sample of non-tumor DNA, respectively, (b) without pre-amplifying the tumor or non-tumor DNA, sequencing a plurality of microsatellite loci from both the tumor and non-tumor DNA using OS-seq to provide sequence reads, wherein the sequenced microsatellite loci comprise mononucleotide, dinucleotide, trinucleotide and tetranucleotide microsatellites loci, and (c) comparing the results.

Method for determining if a tumor has a mutation in a microsatellite

A population of nucleic acid adaptors is provided. In some embodiments, the population contains at least 50,000 different molecular barcode sequences, where the barcode sequences are double-stranded and at least 90% of the barcode sequences have an edit distance of at least 2. In certain cases, the adaptor may have an end in which the top and bottom strands are not complementary (i.e., may be in the form of a Y-adaptor). In some embodiments and depending on the how the adaptor is going to be employed, the other end of the adaptor may have a ligatable end or may be a transposon end sequence.

Robust quantification of single molecules in next-generation sequencing using non-random combinatorial oligonucleotide barcodes

A method for analyzing short tandem repeats (STRs) is described herein. In some embodiments, the method comprises: separately digesting two portions of a genomic sample at sites that are upstream and downstream of an STR; fragmenting those products; ligating adaptors to the fragmentation products; selectively amplifying part of the top strand but not the bottom strand of the ligation products derived from the first portion, and part of the bottom strand but not the top strand of the ligation products derived from the second portion; sequencing at least some of the amplification products to produce a plurality of top strand reads and a plurality of bottom strand reads; and counting the number of STR repeats in a sequence read. A kit for performing the method is also provided.

High resolution STR analysis using next generation sequencing

Methods and reagents for performing digital PCR for detection and quantification of mutant alleles and copy number variation are disclosed. In particular, the invention relates to methods using a nonspecific DNA-binding dye, which produces a fluorescent signal that increases in intensity according to the number of base-pairs present in the PCR amplicon product. The method utilizes mutant-specific and wild-type-specific primers having non-complementary 'tail' sequences of different lengths. Accordingly, the amplicons for the wild-type and mutant alleles differ in length and can be distinguished based on the difference in the intensities of their fluorescent signals. The methods of the invention can be used to detect rare genetic events, including single nucleotide mutations, alterations of copy number, and deletions or insertions of nucleotides.

Quantification of mutant alleles and copy number variation using digital PCR with nonspecific DNA-binding dyes

Certain embodiments provide a method for capturing a genomic fragment. The method may comprise: obtaining a substrate comprising a first population of surface-bound oligonucleotides and a second population of surface-bound oligonucleotides; hybridizing a first member of the first population of surface-bound oligonucleotides to a selection oligonucleotide comprising a region that hybridizes with the first member and a region that contains a genomic sequence; extending the first member of the first population of surface-bound oligonucleotides to produce a support-bound selection primer that comprises a sequence that is complementary to the genomic sequence; hybridizing the support-bound selection primer to a nucleic acid fragment comprising the genomic sequence; extending the support-bound selection primer to produce an extension product that contains a sequence that flanks the genomic sequence, e.g., in a genome; and amplifying the extension product on the substrate.

Direct capture, amplification and sequencing of target DNA using immobilized primers

Provided herein is a method for sample analysis. In some embodiments, the method may involve: a) enzymatically attaching a reactive group to nucleic acid molecules in a sample; b) covalently reacting the reactive group with surface exposed reactive sites on a porous support, thereby covalently tethering the nucleic acid molecules to the porous support; c) performing a primer extension reaction using the tethered nucleic acid molecules as a template to produce primer extension products; and d) eluting the primer extension products from the porous support, while leaving the tethered nucleic acid molecules tethered to the porous support.

Growing community of inventors

Stanford, CA, United States of America

Hanlee P Ji