Enrichment of short nucleic acid fragments in sequencing library preparation

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

(i) ligating double-stranded 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 oligonucleotides less than 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;

(iv) enriching the amplified adapter-fragment constructs for adapter-fragment constructs derived from dsDNA fragments less than 150 bp in length to generate an enriched sample comprising enriched adapter-fragment constructs;

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

(vi) 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.

2. The method according to claim 1 , wherein the test sample comprises 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.

3. The method according to claim 1 , wherein the double-stranded DNA (dsDNA) fragments comprise cell-free DNA (cfDNA) fragments.

4. The method according to claim 1 , wherein the test sample comprises 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.

5. The method according to claim 1 , wherein more than 25% of the dsDNA fragments in the test sample are less than 150 bp in length, prior to the first nucleic acid extension reaction.

6. The method according to claim 1 , wherein the test sample comprises a plasma sample obtained from a patient known to have, or suspected of having cancer.

7. The method according to claim 6 , wherein the test sample comprises nucleic acids originating from healthy cells and from cancer cells.

8. The method according to claim 1 , wherein the dsDNA fragments are purified from the test sample, prior to preparing the enriched sequencing library.

9. The method according to claim 1 , wherein the dsDNA fragments in the test sample or the adapter-fragment constructs derived from the dsDNA fragments in the test sample are enriched for dsDNA fragments or adapter-fragment constructs derived from dsDNA fragments less than 150 bp in length using gel electrophoresis or size selection beads.

10. The method according to claim 9 , wherein the size selection beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, having a length of less than 140 bp.

11. The method according to claim 10 , wherein the size selection beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from dsDNA fragments, having a length that ranges from 60 bp to 140 bp.

12. The method according to claim 1 , wherein the enriched sequencing library is sequenced to generate a plurality of sequence reads.

13. The method according to claim 12 , wherein the sequence reads are identified based on alignment of the sequence reads to a reference genome, or a portion of a reference genome, or based on a de novo assembly.

14. The method according to claim 12 , 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.

15. The method according to claim 14 , wherein monitoring cancer progression further comprises monitoring disease progression, monitoring therapy, or monitoring cancer growth.

16. The method according to a claim 14 , wherein the cancer classification comprises determining cancer type and/or cancer tissue of origin.

17. The method according to claim 14 , wherein the cancer comprises a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, a blastoma, a germ cell tumor, or any combination thereof.

18. The method according to claim 17 , wherein the cancer is selected from the group consisting of: adenocarcinoma, squamous cell carcinoma, 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, breast carcinoma, osteosarcoma, chondrasarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelial sarcoma (mesothelioma), fibrosarcoma, angiosarcoma, liposarcoma, glioma, astrocytoma, myelogenous, granulocytic, lymphatic, lymphocytic, lymphoblastic leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, or any combination thereof.