# US 12,601,010 B2 · Method and kit for the generation of DNA libraries for massively parallel sequencing

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- Number: US 12,601,010 B2
- Document ID: US12601010B2
- Status: Granted
- Kind: B2
- Application number: 18/506,751
- Filed: 2023-11-10
- Published: 2024-05-16
- Granted: 2026-04-14
- Earliest claimed: 2017-07-21
- Assignees: Menarini Silicon Biosystems S.p.A.
- Applicants: Menarini Silicon Biosystems S.p.A.
- Inventors: Valentina Del Monaco; Nicolò Manaresi; Genny Buson; Paola Tononi
- Web page: https://patentgrep.com/patent/US12601010B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

There is disclosed a method of generating a massively parallel sequencing library comprising the steps of :a) providing a primary WGA DNA library (pWGAlib), including fragments comprising a WGA library universal sequence adapter; b) performing a single PCR cycle on the pWGAlib using a first primer (1PR) comprising from 5′ to 3′ a first sequencing adapter (1PR5SA) and a first primer 3′ section (1PR3S) hybridizing to the reverse complementary of the WGA library universal sequence adapter; c) performing a single PCR cycle on the on the product of step b) using a second primer (2PR) comprising from 5′ to 3′ a second sequencing adapter (2PR5SA) different from the 1PR5SA, and a second primer 3′ section (2PR3S) hybridizing to the WGA library universal sequence adapter reverse complementary; d) amplifying by PCR the product of step c) using a third primer comprising the 1PR5SA and a fourth primer comprising 2PR5SA.

## Claims

### Claim 1

A method for low-pass whole genome sequencing comprising the steps of:
generating a plurality of barcoded, massively parallel sequencing libraries comprising the steps of:
(a) providing a primary whole genome amplification (WGA) DNA library (pWGAlib) including fragments comprising a known 5′ sequence section (5SS), a middle sequence section (MSS), and a known 3′ sequence section (3SS) reverse complementary to the known 5′ sequence section, the known 5′ sequence section (5SS) comprising a WGA library universal sequence adapter, and the middle sequence section (MSS) comprising at least an insert section (IS), corresponding to a DNA sequence of the original unamplified DNA prior to WGA:
(b) performing a PCR cycle consisting of a single round of PCR on the primary WGA DNA library using at least one first primer (1PR) comprising at least a first primer 5′ section (1PR5S) and a first primer 3′ section (1PR3S), the first primer 5′ section (1PR5S) comprising at least one first sequencing adapter (1PR5SA), and the first primer 3′ section (1PR3S) hybridizing to the known 3′ sequence section (3SS), so as to obtain a first primer extended WGA DNA library:
(c) performing a PCR cycle consisting of a single round of PCR on the first primer extended WGA DNA library using at least one second primer (2PR) comprising a second primer 5′ section (2PR5S) and a second primer 3′ section (2PR3S), the second primer 5′ section (2PR5S) comprising at least one second sequencing adapter (2PR5SA) different from the at least one first sequencing adapter (1PR5SA), and the second primer 3′ section (2PR3S) hybridizing to the known 3′ sequence section (3SS), so as to obtain a first and second primer extended WGA DNA library:
(d) amplifying by PCR the first primer extended by WGA DNA library and the second primer extended WGA DNA library using at least one third primer (3PR) comprising the first sequencing adapter (1PR5SA) and at least one fourth primer (4PR) comprising the second sequencing adapter (2PR5SA), so as to obtain an amplified first and second primer extended WGA DNA libraries, wherein the first primer 5′ section (1PR5S) further comprises at least one first sequencing barcode (1PR5BC), in 3′ position of the at least one first sequencing adapter (1PR5SA) and in 5′ position of the first primer 3′ section (1PR3S) and/or the second primer 5′ section (2PR5S) further comprises at least one second sequencing barcode (2PR5BC), in 3′ position of the at least one second sequencing adapter (2PR5SA) and in 5′ position of the second primer 3′ section (2PR3S);
providing the plurality of barcoded, massively-parallel sequencing libraries and pooling samples obtained using different sequencing barcodes (BC); and
sequencing the pooled library.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the at least one first primer (1PR) further comprises at least one read sequencing primer sequence (1PRSEQ) in 3′ position of the first primer 5′ section (1PR5S) and in 5′ position of the first primer 3′ section (1PR3S).

### Claim 3 (depends on 1)

The method according to claim 1, further comprising a step of purifying the first primer extended WGA DNA library after step b and/or further comprising a step of purifying the first and second primer extended WGA DNA library after step c and/or further comprising a step of purifying the amplified first and second primer extended WGA DNA library after step d.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the WGA library universal sequence adapter is a DRS-WGA library universal sequence adapter or a MALBAC library universal sequence adapter.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the WGA library universal sequence adapter is a DRS-WGA library universal sequence adapter.

### Claim 6 (depends on 4)

The method according to claim 4, wherein the DRS-WGA library universal sequence adapter is SEQ ID NO:50 and the MALBAC library universal sequence adapter is SEQ ID NO:51.

### Claim 7 (depends on 1)

The method for low-pass whole genome sequencing according to claim 1, wherein the step of pooling samples using different sequencing barcodes (BC) further comprises the steps of:
quantitating the DNA in each of the barcoded, massively-parallel sequencing libraries;
normalizing the amount of barcoded, massively-parallel sequencing libraries.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the middle sequence section (MSS) comprises a flanking 5′ intermediate section (F5) and/or a flanking 3′ intermediate section (F3).