Methods for next generation genome walking and related compositions and kits

Patent metadata

Field	Value
Publication number	US-10988802-B2
Application number	US-201615575711-A
Country	US
Kind code	B2
Filing date	May 20, 2016
Priority date	May 22, 2015
Publication date	Apr 27, 2021
Grant date	Apr 27, 2021

Abstract

Methods are provided herein for identifying rare and/or unknown DNA sequences by next-generation sequencing approaches. Isolated double-stranded (ds), single-stranded (ss), or ds/ss DNA is fragmented and the fragments are polished, phosphorylated, and tailed, as necessary. Fragmentation can be enzymatic or mechanical. A universal adapter sequence is ligated to each fragment, wherein the adapter can have a top strand without a 5′ phosphate, a 3′ with an —H in place of the —OH, and/or a 3′ extra base complementary to any base added to the polished fragments. The ligatamers may then serve as templates for amplification using a forward primer complementary to the adapter sequence and a reverse primer targeted to the fragment sequence. Compositions produced by these methods and kits adapted for performing these methods are also described herein.

First claim

What is claimed is:

1. A composition comprising a plurality of polynucleotides that form a template molecule, each of the plurality of polynucleotides comprising a randomly fragmented native sequence and a universal adapter sequence, the randomly fragmented native sequence comprising a sequence of interest and a native sequence priming domain, the universal adapter sequence comprising 5′ to 3′ a non-primable adapter priming domain and, optionally, a barcode domain consisting of 1 to 20 nucleotides, wherein the universal adapter sequence is located a fixed distance from the 5′ end of the sequence of interest, and wherein the universal adapter sequence has a top strand and a non-extendable bottom strand, where the bottom strand is non-complementary to a primer sequence of the top strand.

2. The composition according to claim 1 , wherein the nucleotide sequence between the universal adapter sequence and the sequence of interest defines an identification sequence.

3. The composition according to claim 2 , wherein when the barcode domain is present, the identification sequence includes the barcode domain.

4. The composition according to claim 1 , further comprising a universal primer whose nucleotide sequence is complementary to the adapter priming domain.

5. The composition according to claim 1 , further comprising a primer whose nucleotide sequence is complementary to a region of the native sequence.

6. The composition according to claim 1 , wherein the sequence of interest is a mutation.

7. The composition according to claim 1 , wherein the sequence of interest is a single nucleotide polymorphism (SNP).

8. The composition according to claim 1 , wherein the sequence of interest is an insertion or deletion (INDEL).

9. The composition according to claim 1 , wherein the adapter priming domain is chosen from those listed in Table 4 and wherein the probability of any given plurality being molecularly unique is greater than 95%, greater than 99%, or greater than 99.9%.

Assignees

• Sigma Aldrich Co Llc

Inventors

• Ward Brian

Classifications

• C40B20/04

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title