# US 2018/0025111 A1 · Strategies for high throughput identification and detection of polymorphisms

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- Number: US 2018/0025111 A1
- Document ID: US20180025111A1
- Status: Published application
- Kind: A1
- Application number: 15/729,328
- Filed: 2017-10-10
- Published: 2018-01-25
- Earliest claimed: 2005-06-23
- Assignees: Keygene N.V.
- Applicants: Keygene N.V.
- Inventors: Michael Josephus Theresia VAN EIJK; Henricus Johannes Adam VAN DER POEL
- Web page: https://patentgrep.com/patent/US20180025111A1

## CPC

- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- C12Q 2600/156: Polymorphic or mutational markers
- C40B 30/10: by measuring physical properties, e.g. mass
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The invention relates to a method for identifying one or more polymorphisms in nucleic acid samples, comprising: (a) performing a reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein the reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using one or more primers to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence to indicate origin of each library obtained by the reproducible complexity reduction; (b) combining the plurality of libraries to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences; (c) aligning the sequences to obtain an alignment; and (d) identifying one or more polymorphisms in the plurality of nucleic acid samples.

## Claims

### Claim 1

A method for identifying genetic variation in nucleic acid samples, comprising:
(a) performing a reproducible complexity reduction on a plurality of nucleic acid samples, wherein the reproducible complexity reduction comprises enrichment using biotinylated capture oligonucleotide hybridization probes to capture a subset of fragments of the nucleic acid samples, and amplifying these fragments using one or more primers to obtain a pool of amplified fragments, wherein the amplified fragments are tagged with a unique identifier sequence;
(b) sequencing at least a portion of the pool of the amplified fragments to obtain sequences;
(c) aligning the sequences obtained in step (b) to obtain an alignment; and
(d) identifying genetic variation in the plurality of nucleic acid samples.

### Claim 2 (depends on 1)

The method of claim 1, wherein the genetic variation is a single nucleotide polymorphism (SNP), a small insertion or a deletion, or a variation in microsatellite repeat number.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the sequencing step (b) is performed on a solid support.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acid samples are human nucleic acid samples.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the solid support is a bead.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the sequencing step (b) comprises dideoxy chain-terminating sequencing.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the sequencing step (b) comprises high-throughput sequencing.

### Claim 6 (depends on 3)

The method according to claim 3, wherein the sequencing step (b) comprises:
(i) annealing amplified fragments to beads, each bead annealing with a single amplified fragment;
(ii) emulsifying the beads in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead;
(iii) loading the beads in wells, each well comprising a single bead; and
(iv) generating a pyrophosphate signal.

### Claim 7 (depends on 6)

The method according to claim 6, wherein, preceding the annealing step, sequencing adaptors are ligated to the amplified fragments.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the sequencing adaptors carry a 3′-T overhang.

### Claim 9 (depends on 1)

The method according to claim 1, wherein at least one of the primers is phosphorylated.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the nucleic acid molecules are tagged using amplification or ligation.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the unique identifier sequence indicates, independently from sequences of the nucleic acid samples, sample origin of each amplified fragment.