# US 11,111,545 B2 · Methods for simultaneous amplification of target loci

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- Number: US 11,111,545 B2
- Document ID: US11111545B2
- Status: Granted
- Kind: B2
- Application number: 17/061,877
- Filed: 2020-10-02
- Published: 2021-01-28
- Granted: 2021-09-07
- Earliest claimed: 2010-05-18
- Assignees: Natera, Inc.
- Applicants: Natera, Inc.
- Inventors: Joshua Babiarz; Tudor Pompiliu Constantin; Lane A. Eubank; George Gemelos; Matthew Micah Hill; Huseyin Eser Kirkizlar; Matthew Rabinowitz; Onur Sakarya; Styrmir Sigurjonsson; Bernhard Zimmermann; Johan Baner; Allison Ryan; Milena Banjevic; Zachary Demko
- Web page: https://patentgrep.com/patent/US11111545B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 2600/156: Polymorphic or mutational markers
- 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

## Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

## Claims

### Claim 1

A method for preparing a fraction of DNA useful for analyzing cancer-associated loci from an individual, comprising:
(a) extracting DNA from a biological sample from the individual to obtain a sample of cell-free DNA (cfDNA);
(b) producing a fraction of the DNA extracted in (a) by amplifying at least 10 target loci relating to cancer-associated mutations to obtain amplification products that comprise the at least 10 target loci, wherein the amplifying comprises:
(1) attaching adaptors to the cfDNA to produce adaptor-attached cfDNA, wherein the adaptors comprise universal priming sequences,
(2) a first PCR amplifying the at least 10 target loci comprising contacting the adaptor-attached cfDNA with a first universal primer and with at least 10 non-identical target specific primers in a single reaction volume, and
(3) a second, nested PCR amplifying the at least 10 target loci amplified in (2) comprising contacting the reaction products from (2) with a second universal primer and with at least 10 non-identical inner target specific primers in a single reaction volume, wherein the at least 10 target loci are single nucleotide variant (SNV) loci; and
(c) analyzing the at least 10 target loci in the fraction of DNA produced in (b).

### Claim 2 (depends on 1)

The method according to claim 1, wherein the analysis comprises correction for allele amplification bias, ambient contamination, and genotype contamination.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the analysis comprises correction for allele amplification bias.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the analysis comprises high throughput sequencing of a plurality of copies of amplicons produced in (b) by a multiplex amplification reaction performed under limiting primer conditions.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the high throughput sequencing comprises sequencing a plurality of copies of a series of amplicons generated from a multiplex amplification reaction, and wherein each amplicon of the series of amplicons spans at least one single nucleotide variant loci of the at least 10 target loci.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the analysis comprises determining whether a single nucleotide variant is present in the sample, comprises identifying a confidence value for each allele determination at each of the at least 10 target loci based at least in part on a depth of read for the loci.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the at least 10 target loci comprises a plurality of the single nucleotide variant sites identified in the TCGA and COSMIC data sets.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the analysis comprises high throughput sequencing of the fraction of DNA produced in (b), and wherein the high throughput sequencing is performed with a depth of read for the at least 10 target loci of at least 100,000, and is capable of detecting a single nucleotide variant with a limit of quantification of 0.1% of the copies of that loci in the sample.

### Claim 9 (depends on 1)

The method according to claim 1, comprising generating nucleic acid sequence data for the at least 10 target loci known to be associated with cancer.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the cfDNA is from breast cancer, lung cancer, colorectal cancer, or ovarian cancer.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the biological sample comprises blood, serum, or plasma.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the method comprises blunting ending and dA-tailing the DNA extracted in (a) prior to attaching adaptors.

### Claim 13 (depends on 1)

The method according to claim 1, wherein step (b) comprises amplifying at least 20 target loci relating to cancer-associated mutations to obtain amplification products.

### Claim 14 (depends on 1)

The method according to claim 1, wherein step (b) comprises amplifying at least 50 target loci relating to cancer-associated mutations to obtain amplification products.

### Claim 15 (depends on 1)

The method according to claim 1, wherein step (b) comprises amplifying at least 100 target loci relating to cancer-associated mutations to obtain amplification products.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the fraction of DNA produced in (b) are tagged with molecular barcodes.

### Claim 17 (depends on 1)

The method according to claim 1, wherein step (c) comprises high-throughput sequencing of the fraction of DNA derived from multiple samples in a single sequencing lane.

### Claim 18 (depends on 1)

The method according to claim 1, wherein at least one of the target specific primers comprises a tail, wherein the tail has no homology to the target loci and comprises a common priming site.

### Claim 19 (depends on 1)

The method according to claim 1, wherein at least one of the target specific primers comprises a priming site for a subsequent amplification to add barcode sequences for multiplex sequencing.