Compositions and methods for evaluating genomic alterations

Patent metadata

Field	Value
Publication number	US-12473589-B2
Application number	US-202519044599-A
Country	US
Kind code	B2
Filing date	Feb 3, 2025
Priority date	Jun 11, 2018
Publication date	Nov 18, 2025
Grant date	Nov 18, 2025

Abstract

Compositions and methods of evaluating genomic alterations in a sample are disclosed.

First claim

What is claimed is:

1 . A method of analyzing a sample, comprising:
(a) contacting a sample comprising first fragments (F1s) having a first target nucleic acid sequence and second fragments (F2s) having a second target nucleic acid sequence with plurality of target capture reagents, said plurality of target capture reagent comprising first target capture reagents (R1s) and second target capture reagents (R2s) to form a plurality of first fragment/first target capture reagent (F1/R1) hybrids and a plurality of second fragment/second target capture reagent (F1/R2) hybrids,
wherein each R1 comprises an oligonucleotide comprising a first nucleic acid sequence that hybridizes to an F1 from the F1s, the oligonucleotides of the R1s are the same, a first portion of the R1s comprises a first member of a binding pair, and a second portion of the R1s lacks the first member of the binding pair, and
wherein each R2 comprises an oligonucleotide comprising a second nucleic acid sequence that hybridizes to an F2 from the F2s, the oligonucleotides of the R2s are the same, a first portion of the R2s comprises the first member of a binding pair, and a second portion of the R2s lacks the first member of the binding pair;
(b) contacting the F1/R1 hybrids with a substrate comprising a second member of the binding pair that binds to the first member of the binding pair to form F1/R1 hybrid/substrate complexes, and contacting the F2/R2 hybrids with the substrate to form F2/R2 hybrid/substrate complexes, wherein the proportion of F1/R1 hybrids that bind to the substrate is greater than the proportion of F2/R2 hybrids that bind to the substrate; and (c) analyzing first fragments from the plurality of F1/R1 hybrids and second fragments from the F2/R2 hybrids.

2 . The method of claim 1 , wherein F1 comprises a sequence associated with a phenotype, treatment outcome, diagnosis, or prognosis.

3 . The method of claim 1 , wherein the proportion of R1s that comprises the first member of the binding pair is greater than the proportion of R2s that comprises the first member of the binding pair.

4 . The method of claim 1 , wherein analyzing the first fragment comprises:
sequencing a subgenomic interval sequence within the first fragments at a depth of at least 5,000× depth; or sequencing a subgenomic interval sequence within the second fragments at a depth of 800× to less than 5,000×.

5 . The method of claim 1 , further comprising:
contacting a plurality of third fragment/third target capture reagent (F3/R3) hybrids with a substrate to form F3/R3 hybrid/substrate complexes, wherein R3s comprise R3s that comprise a first member of a binding pair and R3s that lack the first member of the binding pair; and analyzing third fragments from the plurality of F3/R3 hybrids.

6 . The method of claim 1 , wherein the sample comprises genomic DNA, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or RNA.

7 . The method of claim 1 , wherein the contacting occurs in solution.

8 . The method of claim 1 , wherein the contacting occurs on a solid surface.

9 . The method claim 1 , wherein the first member of the binding pair comprises a biotin moiety, and wherein the second member of the binding pair comprises a streptavidin moiety, an avidin moiety, or a modified version thereof.

10 . The method of claim 1 , wherein the first member of the binding pair in R1 is coupled to a moiety that captures F1 via a linker, and wherein the first member of the binding pair in R2 is coupled to a moiety that captures F2 via a linker.

11 . The method of claim 1 , wherein analyzing the first fragment and the second fragment comprises sequencing at least a portion of F1 from the plurality of F1/R1 hybrid/substrate complexes, and sequencing at least a portion of F2 from the plurality of F2/R2 hybrid/substrate complexes.

12 . The method of claim 11 wherein F1 is sequenced to a greater depth than F2.

13 . The method of claim 1 , further comprising contacting a library with a first target capture reagent (R1) and a second target capture reagent (R2) to provide the F1/R1 hybrids and the F2/R2 hybrids.

14 . The method of claim 13 , further comprising acquiring a read for a subject interval comprising an alteration from the first fragment or the second fragment.

15 . The method of claim 13 , comprising acquiring reads for subject intervals in a plurality of genes.

16 . The method of claim 15 , wherein the plurality of genes comprises associated with cell division, cell growth, cell survival, or cancer.

17 . A method of analyzing a sample, comprising:
(a) contacting a sample comprising first fragments (F1s) having a first target nucleic acid sequence and second fragments (F2s) having a second target nucleic acid sequence with plurality of target capture reagents, said plurality of target capture reagent comprising first target capture reagents (R1s) and second target capture reagents (R2s) to form a plurality of first fragment/first target capture reagent (F1/R1) hybrids and a plurality of second fragment/second target capture reagent (F1/R2) hybrids,
wherein each R1 comprises an oligonucleotide comprising a first nucleic acid sequence that hybridizes to an F1 from the F1s, the oligonucleotides of the R1s are the same, a first portion of the R1s comprises a first member of a binding pair, and a second portion of the R1s lacks the first member of the binding pair,
wherein each R2 comprises an oligonucleotide comprising a second nucleic acid sequence that hybridizes to an F2 from the F2s, the oligonucleotides of the R2s are the same, a first portion of the R2s comprises the first member of a binding pair, and a second portion of the R2s lacks the first member of the binding pair, wherein the proportion of R1s that comprise the first member of the binding pair is greater than the proportion of R2s that comprise the first member of a binding pair, and
wherein the first member of the binding pair is capable of binding to a second member of the binding pair disposed on a substrate;
(b) contacting the plurality of F1/R1 hybrids with the substrate to form F1/R1 hybrid/substrate complexes, and contacting the plurality of F2/R2 hybrids with the substrate to form F2/R2 hybrid/substrate complexes, wherein the proportion of F1/R1 hybrids which bind to the substrate is greater than the proportion of F2/R2 hybrids which bind to the substrate; and (c) sequencing F1 from the plurality of F1/R1 hybrid/substrate complexes, and sequencing F2 from the plurality of F2/R2 hybrid/substrate complexes, wherein F1 is sequenced to a greater depth than F2.

18 . A method of analyzing a sample, comprising:
(1) providing a sample comprising genomic DNA, cell-free DNA (cfDNA), or circulating tumor DNA (ctDNA) from a subject; (2) obtaining or isolating nucleic acids from the sample; (3) fragmenting the nucleic acids to provide a plurality of fragments (Fs); (4) attaching adapter sequences to the plurality of fragments (Fs) to provide a plurality of adapterized fragments (AFs); (5) amplifying a first AF (AF1) to provide a plurality of AF1s, and amplifying a second AF (AF2) to provide a plurality of AF2s; (6) contacting the plurality of AF1s with first target capture reagents (R1s), each R1 comprising an oligonucleotide comprising a first nucleotide sequence that hybridizes to an AF1 from the plurality of AF1s, to provide a plurality of AF1/R1 hybrids, wherein the oligonucleotides of the R1s are the same, a first portion of the R1s comprises a first member of a binding pair, and a second portion of the R1s lacks t

Assignees

• Found Medicine Inc

Inventors

• Otto Geoffrey Alan
• Clark Travis
• Lipson Doron
• Lieber Daniel
• Fabrizio David

Classifications

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title