Enrichment of short nucleic acid fragments in sequencing library preparation

Patent metadata

Field	Value
Publication number	US-11851650-B2
Application number	US-201816146901-A
Country	US
Kind code	B2
Filing date	Sep 28, 2018
Priority date	Sep 28, 2017
Publication date	Dec 26, 2023
Grant date	Dec 26, 2023

Abstract

Methods for preparing enriched sequencing libraries from test samples that contain double-stranded deoxyribonucleic acid (dsDNA) are provided.

First claim

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) 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.

Assignees

• Grail Llc

Inventors

• Jung Byoungsok
• Aravanis Alex

Classifications

• C12N15/1058 Primary

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N15/101

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title