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1. (WO2019008172) TARGET-ENRICHED MULTIPLEXED PARALLEL ANALYSIS FOR ASSESSMENT OF TUMOR BIOMARKERS
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Our ef.: B281-0008WO1 10719 Berlin

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) preferably and optionally each member sequence binds to the tumor biomarker 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 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) performing statistical analysis on the enriched library sequences, optionally utilizing only fragments of a specific size range, to thereby detect the tumor biomarker(s) in the DNA sample.

2. The method of claim 1, 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 tumor biomarker sequence of interest but has different start and/or stop positions with respect to a reference coordinate system for the tumor biomarker sequence of interest.

3. The method of claim 1 or 2, wherein each TACS family comprises at least 3 member sequences.

4. The method of claim 1 to 3, wherein the pool of TACS comprises at least 5 different TACS families.

5. The method of any one of claims 1 to 4, 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 at least 3 base pairs.

6. The method of any one of claims 1 to 4, 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 at least 5 base pairs.

7. The method of any one of claims 1 to 6, wherein each member sequence within the pool of TACS is at least 160 base pairs in length.

8. The method of any one of claims 1 to 7, wherein the GC content of the TACS is between 19% and 46%.

9. The method of any one of claims 1 to 8, wherein amplification of the enriched library is performed in the presence of blocking sequences that inhibit amplification of wild-type sequences.

10. The method of any one of claims 1 to 9, wherein members of the sequencing library that bind to the pool of TACS are partially complementary to the TACS.

11. The method of any one of claims 1 to 10, wherein the pool of TACS is fixed to a solid support.

12. The method of claims 1 to 11, wherein the TACS are biotinylated and are bound to streptavidin-coated magnetic beads.

13. The method of any one of claims 1 to 12, wherein the DNA sample comprises cell free tumor DNA (cftDNA).

14. The method of any one of claims 1 to 12, wherein the DNA sample is selected from a group comprising of a plasma sample, a urine sample, a sputum sample, a cerebrospinal fluid sample, an ascites sample and a pleural effusion sample from subject having or suspected of having a tumor.

15. The method of any one of claims 1 to 12, wherein the DNA sample is from a tissue sample from a subject having or suspected of having a tumor.

16. The method of any of claims 1 to 15, wherein the statistical analysis comprises a segmentation algorithm.

17. The method of claim 16, wherein the segmentation algorithm is selected from the group consisting of likelihood-based segmentation, segmentation using small overlapping windows, segmentation using parallel pairwise testing, and combinations thereof.

18. The method of any one of claims 1 to 15, wherein the statistical analysis comprises a score- based classification system.

19. The method of any one of claims 1 to 15, wherein sequencing of the enriched library provides a read-depth for the genomic sequences of interest and read-depths for reference loci and the statistical analysis comprises applying an algorithm that tests sequentially the read-depth of the loci of from the genomic sequences of interest against the read-depth of the reference loci, the algorithm comprising steps for: (a) removal of inadequately sequenced loci; (b) GC-content bias alleviation; and (c) genetic status determination.

20. The method of claim 19, wherein GC-content bias is alleviated by grouping together loci of matching GC content.

21. The method of any one of claims 1 to 15, wherein sequencing of the enriched library provides the number and size of sequenced fragments for TACS-specific coordinates and the statistical analysis comprises applying an algorithm that tests sequentially the fragment-size proportion for the genomic sequence of interest against the fragment-size proportion of the reference loci, the algorithm comprising steps for: (a) removal of fragment-size outliers; (b) fragment- size proportion calculation; and (c) genetic status determination.

22. The method of any one of claims 1 to 21, wherein the pool of TACS binds to a plurality of tumor biomarker sequences of interest selected from a group comprising AKT1, ALK, APC, A , ARAF, ATM, BAP1, BARD1, BMPR1A, BRAF, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (pl4ARF), CDKN2A (pl6INK4a), CHEK2, CTNNB1, DDB2, DDR2, DICERl, EGFR, EPCAM, ERBB2, ERBB3, ERBB4, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXW7, FGFR1, FGFR2, FLT3, FOXA1, FOXL2, GATA3, GNA11, GNAQ, GNAS, GREM1, HOXB13, IDH1, IDH2, JAK2, KEAP1, KIT, KRAS, MAP2K1, MAP3K1, MEN1, MET, MLH1, MPL, MRE11A, MSH2, MSH6, MTOR, MUTYH, MYC, MYCN, NBN, NPM1, NRAS, NTRK1, PALB2, PDGFRA, PIK3CA, PIK3CB, PMS2, POLD1, POLE,

POLH, PTEN, AD50, RAD51C, RAD51D, RAF1, RBI, RET, ROS1, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SLX4, SMAD4, SMARCA4, SPOP, STAT, STK11, TMPRSS2, TP53, VHL, XPA, XPC and combinations thereof.

23. The method of any one of claims 1 to 21, wherein the pool of TACS binds to a plurality of tumor biomarker sequences of interest selected from a group comprising EGFR_6240, KRAS_521, EGFR_6225, NRAS_578, NRAS_580, PIK3CA_763, EGFR_13553, EGFR_18430, BRAF_476, KIT_1314, NRAS_584, EGFR_12378, and combinations thereof.

24. The method of any one of claims 1 to 23, which further comprises making a diagnosis of the subject based on detection of at least one tumor biomarker sequence.

25. The method of any one of claims 1 to 24, which further comprises selecting a therapeutic regimen for the subject based on detection of at least one tumor biomarker sequence.

26. The method of any one of claims 1 to 25, which further comprises monitoring treatment efficacy of a therapeutic regimen in the subject based on detection of at least one tumor biomarker sequence.

27. A kit for performing the method of claim 1, wherein the kit comprises a container comprising the pool of TACS and instructions for performing the method, 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) preferably and optionally each member sequence binds to a tumor biomarker 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.

28. The kit of claim 27, wherein the pool of TACS comprises a plurality of TACS families, wherein each TACS family comprises a plurality of member sequences, wherein each member sequence binds to the same tumor biomarker sequence of interest but has different start and/or stop positions with respect to a reference coordinate system for the tumor biomarker sequence of interest.