US 12,270,069 B2
Enrichment of targeted genomic regions for multiplexed parallel analysis
George Koumbaris, Lithrodontas (CY); Marios Ioannides, Nicosia (CY); Elena Kypri, Nicosia (CY); Acilleas Achilleos, Limassol (CY); Petros Mina, Nicosia (CY); Kyriakos Tsangaras, Limassol (CY); and Philippos Patsalis, Nicosia (CY)
Assigned to MEDICOVER PUBLIC CO LTD, Nicosia (CY)
Appl. No. 16/625,428
Filed by MEDICOVER PUBLIC CO LTD, Nicosia (CY)
PCT Filed Jul. 6, 2018, PCT No. PCT/EP2018/068402
§ 371(c)(1), (2) Date Dec. 20, 2019,
PCT Pub. No. WO2019/008148, PCT Pub. Date Jan. 10, 2019.
Claims priority of provisional application 62/529,667, filed on Jul. 7, 2017.
Prior Publication US 2020/0157602 A1, May 21, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12N 15/10 (2006.01); C12Q 1/6886 (2018.01); G06F 17/18 (2006.01); G16B 30/00 (2019.01)
CPC C12Q 1/6806 (2013.01) [C12N 15/1093 (2013.01); C12Q 1/6886 (2013.01); G06F 17/18 (2013.01); G16B 30/00 (2019.02); C12Q 2525/185 (2013.01); C12Q 2525/204 (2013.01); C12Q 2535/122 (2013.01); C12Q 2537/159 (2013.01); C12Q 2600/156 (2013.01)] 18 Claims
 
1. A method of testing for risk of a genetic abnormality in a DNA sample comprising genomic sequences of interest, the method comprising:
(a) preparing a sequencing library from the DNA sample;
(b) hybridizing the sequencing library to a pool of double-stranded TArget Capture Sequences (TACS), wherein the pool of TACS comprises a plurality of TACS families directed to different genomic sequences of interest, wherein each TACS family comprises a plurality of member sequences, wherein each member sequence binds to the same genomic sequence of interest but has different start and/or stop positions with respect to a reference coordinate system for the genomic sequence of interest, and further wherein:
(i) each member sequence within each TACS family is between 100-500 base pairs in length, each member sequence having a 5′ end and a 3′ end;
(ii) each member sequence binds to the same genomic sequence of interest at least 50 base pairs away, on both the 5′ end and the 3′ end, from regions harboring Copy Number Variations (CNVs), Segmental duplications or repetitive DNA elements; and
(iii) the GC content of the pool of TACS is between 19% and 80%, as determined by calculating the GC content of each member within each family of TACS;
(c) isolating members of the sequencing library that bind to the pool of TACS to obtain an enriched library;
(d) amplifying and sequencing the enriched library; and
(e) performing statistical analysis on the enriched library sequences to thereby determine risk of a genetic abnormality in the DNA sample;
wherein the genomic sequences of interest comprise DNA fragments, and
wherein the start and/or stop positions for the member sequences within a TACS family, with respect to a reference coordinate system for the genomic sequence of interest, are staggered by 5 to 10 base pairs.