Patent ID: 11859249
Assignee: MENARINI SILICON BIOSYSTEMS S.P.A.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method of generating a massively parallel sequencing library 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 produced by step (b) 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 primer extended WGA DNA library and a second primer extended WGA DNA library; and,
(d) amplifying by PCR the first primer extended 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.