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1. (WO2019067973) ENRICHMENT OF SHORT NUCLEIC ACID FRAGMENTS IN SEQUENCING LIBRARY PREPARATION
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WHAT IS CLAIMED IS:

1. A method for preparing an enriched sequencing library, the method comprising:

(a) obtaining a test sample comprising a plurality of double-stranded deoxyribonucleic acid (dsDNA) fragments;

(b) ligating double-strand DNA (dsDNA) adapters to both ends of the dsDNA fragments to generate a plurality of adapter-fragment constructs, wherein the dsDNA adapter comprises a first strand and a second strand;

(c) amplifying the adapter-fragment constructs to generate a sequencing library, wherein the adapter-fragment constructs are amplified in the presence of a blocker, the blocker comprising an oligonucleotide sequence having sequence complementarity with at least a portion of the 5 '-end of the first strand and at least a portion of the 3 '-end of the second strand; and

(d) enriching the sequencing library for adapter-fragment constructs derived from dsDNA fragments less than about 150 bp in length to generate an enriched sequencing library.

2. A method for preparing an enriched sequencing library, the method comprising:

(a) obtaining a test sample comprising a plurality of double-stranded deoxyribonucleic acid (dsDNA) fragments;

(b) preparing an enriched sequencing library from the test sample, wherein preparing the sequencing library comprises:

(i) ligating double-strand DNA adapters to both ends of the dsDNA fragments to generate a plurality of adapter-fragment constructs;

(ii) adding a first set of primers to the adapter-fragment constructs, wherein the first set of primers comprise single-stranded oligonucleotide less than about 50 nucleotides in length;

(iii) hybridizing the first set of primers to the adapter-fragment constructs and extending the first set of primers in a first nucleic acid extension reaction using a polymerase to generate a plurality of amplified adapter- fragment constructs;

(iii) enriching the amplified adapter-fragment constructs for adapter- fragment constructs derived from dsDNA fragments less than about 150 bp in

length to generate an enriched sample comprising enriched adapter-fragment constructs;

(iv) adding a second set of primers to the enriched sample, wherein the second set of primers comprise single-stranded oligonucleotide greater than about 50 nucleotides in length; and

(v) hybridizing the second set of primers to the enriched adapter- fragment constructs and extending the second set of primers in a second nucleic acid extension reaction using a polymerase to generate a sequencing library.

3. A method for preparing an enriched sequencing library, the method comprising:

(a) obtaining a test sample comprising a plurality of double-stranded deoxyribonucleic acid (dsDNA) fragments;

(b) ligating double-strand DNA (dsDNA) adapters to both ends of the dsDNA fragments to generate a plurality of adapter-fragment constructs, wherein the dsDNA adapters are shorter than 50 nucleotides in length;

(c) amplifying the plurality of adapter-fragment constructs to generate a sequencing library;

(d) incubate the sequencing library at from about 45°C to about 70°C for from about 2 min to about 60 min, wherein said incubation step denatures adapter dimers; and

(e) enriching the sequencing library for adapter-fragment constructs derived from dsDNA fragments less than about 150 bp in length to generate an enriched sequencing library.

4. The method according to any one of the preceding claims, wherein the test sample comprising a plurality of dsDNA fragments synthesized from single-stranded ribonucleic acid (ssRNA) molecules, wherein synthesizing the dsDNA fragments from ssRNA molecules comprises:

(a) obtaining a test sample comprising a plurality of single-stranded ribonucleic acid (ssRNA) molecules;

(b) adding an RNA primer to the ssRNA test sample and extending the RNA primer in a first nucleic acid extension reaction using reverse transcriptase to generate a plurality of complementary DNA (cDNA) sequences, wherein the cDNA sequences are complementary to the one or more RNA templates; and

(c) adding one or more DNA primers to the reaction mixture and extending the one or more DNA primers in a second nucleic acid extension reaction using a DNA polymerase to generate a plurality of dsDNA fragments.

5. The method according to any preceding claim, wherein the double-stranded DNA (dsDNA) fragments are cell-free DNA (cfDNA) fragments.

6. The method according to any preceding claim, wherein the test sample is selected from whole blood, a blood fraction, plasma, serum, urine, fecal, saliva, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, peritoneal fluid, or any combination thereof.

7. The method according to any preceding claim, wherein the adapter-fragment constructs derived from dsDNA fragments less than about 150 bp in length comprises more than 25% of the dsDNA molecules in the test sample.

8. The method according to any preceding claim, wherein the test sample is a plasma sample obtained from a patient known to have, or suspected of having cancer.

9. The method according to claim 8, wherein the test sample includes nucleic acids originating from healthy cells and from cancer cells.

10. The method according to any preceding claim, wherein the dsDNA fragments are purified from the test sample, prior to ligation step (c).

11. The method according to any one of the preceding claims, wherein dsDNA fragments are enriched using gel electrophoresis or size selection beads.

12. The method according to claim 11, wherein the size selection beads are solid phase reversible immobilization (SPRI) beads.

13. The method according to claim 1 1, wherein the SPRI beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, having a length of less than 140 bp in length.

14. The method according to claim 11, wherein the SPRI beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, having a length of less than 120 bp in length.

15. The method according to claim 11, wherein the SPRI beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, having a length of less than 100 bp in length.

16. The method according to claim 12, wherein the SPRI beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, from about 60 bp to about 140 bp in length.

17. The method according to claim 12, wherein the SPRI beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, from about 80 bp to about 120 bp in length.

18. The method according to any preceding claim, wherein the dsDNA fragments are modified prior to ligation of the double-stranded DNA adapters.

19. The method according to claim 18, wherein the modification comprises end-repairing, A-tailing, phosphorylation, or any combination thereof.

20. The method according to claim 1, wherein adapter-fragment constructs are amplified in the presence of a single-stranded DNA oligonucleotide blocker, and wherein the blocker includes a 3 '-end blocking nucleotide prohibiting extension of the blocker during amplification.

21. The method according to any of the preceding claims, wherein the enriched sequencing library is sequenced to generate a plurality of sequence reads.

22. The method according to any one of claim 21, wherein the sequence reads are obtained from next-generation sequencing (NGS).

23. The method according to any one of claim 21, wherein the sequence reads are obtained from massively parallel sequencing using sequencing-by-synthesis.

24. The method according to any one of claim 21, wherein the sequence reads are obtained from paired-end sequencing.

25. The method according to any one of claims 21-24, wherein the sequence reads are identified based on alignment of the sequence reads to a reference genome, or a portion of a reference genome.

26. The method according to any one of claims 21-24, wherein the sequence reads are identified based on a de novo assembly.

27. The method according to any one of claims 21-26, wherein the plurality of sequence reads are used for detecting cancer, screening for cancer, determining cancer stage or status, monitoring cancer progression, and/or determining a cancer classification.

28. The method according to claim 27, wherein monitoring cancer progression further comprises monitoring disease progression, monitoring therapy, or monitoring cancer growth.

29. The method according to a claim 27, wherein the cancer classification comprises determining cancer type and/or cancer tissue of origin.

30. The method according to any one of claims 27-29, wherein the cancer comprises a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, a blastoma, a germ cell tumor, or any combination thereof.

31. The method according to claim 30, wherein the carcinoma is an adenocarcinoma.

32. The method according to claim 30, wherein the carcinoma is a squamous cell carcinoma.

33. The method according to claim 30, wherein the carcinoma is selected from the group consisting of: small cell lung cancer, non-small-cell lung, nasopharyngeal, colorectal, anal, liver, urinary bladder, cervical, testicular, ovarian, gastric, esophageal, head-and-neck, pancreatic, prostate, renal, thyroid, melanoma, and breast carcinoma.

34. The method according to claim 30, wherein the sarcoma is selected from the group consisting of: osteosarcoma, chondrasarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelial sarcoma (mesothelioma), fibrosarcoma, angiosarcoma, liposarcoma, glioma, and astrocytoma.

35. The method according to claim 30, wherein the leukemia is selected from the group consisting of: myelogenous, granulocytic, lymphatic, lymphocytic, and lymphoblastic leukemia.

36. The method according to claim 30, wherein the lymphoma is selected from the group consisting of: Hodgkin's lymphoma and Non-Hodgkin's lymphoma.