Enrichment of short nucleic acid fragments in sequencing library preparation
Methods for preparing enriched sequencing libraries from test samples that contain double-stranded deoxyribonucleic acid (dsDNA) are provided.
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-stranded DNA (dsDNA) adapters to both ends of the plurality of dsDNA fragments to generate a plurality of adapter-fragment constructs, wherein each of the dsDNA adapters comprises a first strand and a second strand;
(c) amplifying the plurality of adapter-fragment constructs to generate a sequencing library, wherein the plurality of 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 of the dsDNA adapters; and
(d) enriching the sequencing library for adapter-fragment constructs derived from the plurality of dsDNA fragments less than about 150 bp in length to generate an enriched 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 plurality of 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 test sample comprising the plurality of ssRNA molecules to generate a reaction mixture 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 plurality of cDNA sequences are complementary to the plurality of ssRNA molecules; 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 plurality of 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 matter, saliva, a tissue biopsy, pleural fluid, pericardial fluid, cerebrospinal fluid, peritoneal fluid, or any combination thereof.
5. The method according to claim 1 , wherein more than 25% of the plurality of dsDNA fragments in the test sample are less than 150 bp in length, prior to the amplifying.
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 plurality of dsDNA fragments are purified from the test sample, prior to preparing the enriched sequencing library.
9. The method according to claim 1 , wherein the plurality of dsDNA fragments in the test sample or the plurality of adapter-fragment constructs derived from the plurality of dsDNA fragments in the test sample are enriched for dsDNA fragments or adapter-fragment constructs derived from the plurality of 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 the plurality of dsDNA fragments, having a length of less than 140 bp.
11. The method according to claim 9 , wherein the size selection beads are utilized to enrich for dsDNA fragments, or adapter-fragment constructs derived from the plurality of 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 plurality of sequence reads are identified based on alignment of the plurality of 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 a claim 14 , wherein the cancer classification comprises determining cancer type and/or cancer tissue of origin.
16. 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.
17. The method according to claim 16 , 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, chondrosarcoma, 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.