# US 2022/0033901 A1 · Universal sanger sequencing from next-gen sequencing amplicons

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- Number: US 2022/0033901 A1
- Document ID: US20220033901A1
- Status: Published application
- Kind: A1
- Application number: 17/397,280
- Filed: 2021-08-09
- Published: 2022-02-03
- Earliest claimed: 2012-12-28
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments LLC
- Inventors: Heather Sanders; Hai-Rong Li; Feras Hantash; Frederic Waldman
- Web page: https://patentgrep.com/patent/US20220033901A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2600/16: Primer sets for multiplex assays
- 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

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

## Claims

### Claim at position 1

-20. (canceled)

### Claim 21

A method of generating an amplicon for next generation sequencing (NSG) for at least one region of interest comprising:
(a) adding to a sample of DNA:
(i) a first primer set specific for the region of interest consisting of two primers each comprising a 5′ region encoding an adapter and a 3′ region specific for the region of interest; and
(ii) a second primer set consisting of two primers each of which comprises:
(A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQ ID NO: 1) or AACAGCTATGACCATG (SEQ ID NO: 2);
(B) a spacer comprising at least 10 nucleotides; and
(C) a 3′ adapter that is substantially identical to the adaptor region,
wherein the adapter has a Tm of about 55° C. and the second primer set has a Tm of at least 70° C.; and
(b) performing a plurality of PCR cycles with a first annealing temperature and a second annealing temperature.

### Claim 22 (depends on 21)

The method of claim 21, wherein the first primer set binds with a Tm of about 60° C. immediately upstream and downstream of the region of interest.

### Claim 23 (depends on 21)

The method of claim 21, wherein the adaptor comprises a sequence selected from Table 2.

### Claim 24 (depends on 21)

The method of claim 21, wherein the plurality of PCR cycles comprises about 10 cycles at the first annealing temperature and about 30 cycles at the second annealing temperature.

### Claim 25 (depends on 24)

The method of claim 24, wherein the first annealing temperature is about 55° C.

### Claim 26 (depends on 24)

The method of claim 24, wherein the second annealing temperature is between about 60° C. to about 62° C.

### Claim 27 (depends on 21)

The method of claim 21, wherein the plurality of PCR cycles further comprises:
(i) a heating step at about 95° C.;
(ii) an extending step for about 10 cycles of:
(A) about 95° C.;
(B) about 55° C.; and
(C) about 72° C., and
(iii) an extending step for about 30 cycles of:
(A) about 95° C.;
(B) between about 60° C. to about 62° C.; and
(C) about 72° C.

### Claim 28 (depends on 27)

The method of claim 27, wherein the plurality of PCR cycles further comprises an additional extending step at a temperature of about 75° C. for about 15 minutes.

### Claim 29 (depends on 21)

The method of claim 21, wherein the ratio of the first primer set to the second primer set is greater than 1:1.