Direct-to-library methods, systems, and compositions

Abstract

Provided herein are direct-to-library methods, systems, and compositions.

Description

Our Ref: PT2026002449-FF-P-HK Title of Invention: Direct-to-Library Methods, Systems, and Compositions This document provides direct-to-library methods, systems, and compositions. Abstract

Claims

1. A method of preparing a nucleic acid library from an initial sample, comprising generating a nucleic acid library from the said initial sample; wherein nucleic acids used to generate the nucleic acid library are not extracted from the initial sample before preparing the nucleic acid library; and wherein the nucleic acids used to generate the library comprise RNA.

2. The method of claim 1, further comprising converting the RNA to cDNA.

3. The method of claim 2, wherein the converting is performed via incubation with a reverse transcriptase.

4. The method of claim 1, wherein the nucleic acids used to generate the library further comprise DNA.

5. The method of claim 1, further comprising a polyadenylation process.

6. The method of claim 1, wherein the generating the nucleic acid library comprises: (a) denaturing the nucleic acids to produce denatured nucleic acids, (b) attaching a 3'- and 5'-end adapter concurrently or in succession, (c) enriching adapted nucleic acids, and (d) eluting the adapted nucleic acid strands.

7. The method of claim 6, wherein the generating the nucleic acid library further comprises incubating with a protease prior to denaturing the nucleic acids or amplifying the complementary strands after eluting the nucleic acid strands.

8. The method of claim 7, further comprising converting the RNA to cDNA, wherein the incubating with the protease is performed subsequent to converting the RNA to cDNA.

9. The method of claim 1, wherein the initial sample is from a human subject.

10. The method of claim 1, wherein the initial sample comprises a blood sample, a plasma sample, a serum sample, a synovial fluid sample, a bronchoalveolar lavage sample, a cerebrospinal fluid sample, or a urine sample.

11. The method of claim 1, wherein the initial sample is a plasma sample.

12. The method of claim 6, further comprising immobilizing adapted nucleic acids on beads or columns.

13. The method of claim 12, wherein the beads are magnetic beads.

14. The method of claim 6, wherein attaching the 3'-end adapter to the denatured nucleic acids comprises attaching with a splint oligonucleotide.

15. The method of claim 6, wherein the attaching the 3'- and 5'-end adapter comprises a ligation.