US 12,435,374 B2
Target-enriched multiplexed parallel analysis for assessment of tumor biomarkers
George Koumbaris, Lithrodontas (CY); Marios Ioannides, Nicosia (CY); Elena Kypri, Nicosia (CY); Acilleas Achilleos, Limassol (CY); Petros Mina, Nicosia (CY); Alexia Eliades, Nicosia (CY); Charalambos Loizides, Nicosia (CY); and Philippos Patsalis, Nicosia (CY)
Assigned to NIPD GENETICS PUBLIC COMPANY LIMITED, Nicosia (CY)
Appl. No. 16/625,447
Filed by NIPD GENETICS PUBLIC COMPANY LIMITED, Nicosia (CY)
PCT Filed Jul. 6, 2018, PCT No. PCT/EP2018/068441
§ 371(c)(1), (2) Date Dec. 20, 2019,
PCT Pub. No. WO2019/008172, PCT Pub. Date Jan. 10, 2019.
Claims priority of provisional application 62/529,779, filed on Jul. 7, 2017.
Prior Publication US 2022/0228219 A1, Jul. 21, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6806 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6806 (2013.01)] 14 Claims
 
1. A method of detecting one or more tumor biomarkers in a DNA sample from a subject having or suspected of having a tumor, 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) that bind to one or more tumor biomarker sequences of interest, wherein:
(i) each member sequence within the pool of TACS 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 one or more tumor biomarker sequences 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 the pool 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) selecting one or more fragments from the enriched library, the one or more fragments being at most 160 bp in length, and comparing the one or more fragments from the enriched library with fragments from a reference region, to assign a score value, wherein the score value above a threshold is indicative of the presence of the one or more tumor biomarker(s) in the DNA sample,
wherein the pool of TACS comprises a plurality of TACS families each directed to a different tumor biomarker sequence of interest, wherein each TACS family comprises a plurality of member sequences, wherein each member sequence binds to the same one or more tumor biomarker sequences of interest but has different start and/or stop positions with respect to a reference coordinate system for the one or more tumor biomarker sequences of interest, wherein the start and/or stop positions for each member sequence within a TACS family, with respect to a reference coordinate system for the one or more tumor biomarker sequences of interest, are staggered by 5-10 base pairs.