Sample preparation methods, systems and compositions
The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.
1. A method for concurrent processing of different nucleic acid forms in a sample comprising:
(a) providing a sample comprising a first nucleic acid form and a second nucleic acid form;
(b) denaturing said first nucleic acid form and said second nucleic acid form;
(c) ligating a first adapter to one end of said first nucleic acid form using a first ligase that has a preference for said first nucleic acid form and ligating a second adapter to one end of said second nucleic acid form using a second ligase that has a preference for said second nucleic acid form, wherein said first adapter and said second adapter each comprise an identifying sequence that is different from each other; and
(d) detecting said ligated nucleic acid forms.
2. The method of claim 1 , wherein said first nucleic acid form is DNA.
3. The method of claim 2 , wherein said first nucleic acid form is ssDNA.
4. The method of claim 2 , wherein said DNA is derived from a bacterium.
5. The method of claim 2 , further comprising sequencing said DNA.
6. The method of claim 2 , further comprising identifying sequences associated with said first adapter as originating from said DNA.
7. The method of claim 1 , further comprising identifying sequences associated with said second adapter as originating from said second nucleic acid form.
8. The method of claim 7 , wherein said second nucleic acid form is RNA.
9. The method of claim 1 , wherein said ligating of said first adapter and said ligating of said second adapter occurs concurrently.
10. The method of claim 1 , wherein said ligating of said first adapter and said ligating of said second adapter occurs within a single reaction mixture.
11. The method of claim 8 , further comprising sequencing said RNA.
12. The method of claim 9 , wherein said concurrent ligating of said first adapter and said second adapter is performed in the same reaction tube.
13. The method of claim 1 , wherein said first ligase that has preference for said first nucleic acid form is a DNA ligase.
14. The method of claim 13 , wherein said DNA ligase is PBCV-1 DNA ligase/ Chlorella virus DNA ligase, CircLigase ssDNA ligase, Thermostable DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, E. coli DNA ligase or any combination thereof.
15. The method of claim 13 , wherein said DNA ligase is T4 DNA ligase.
16. The method of claim 13 , wherein said DNA ligase is CircLigase ssDNA ligase.
17. The method of claim 1 , wherein said second ligase that has a preference for said second nucleic acid form is an RNA ligase.
18. The method of claim 17 , wherein said RNA ligase is T4 RNA ligase, T4 RNA ligase 2, T4 RNA ligase 2 Truncated, T4 RNA ligase 2 Truncated K227Q, T4 RNA ligase 2, Truncated KQ, RtcB ligase, CircLigase RNA ligase or any combination thereof.
19. The method of claim 17 , wherein said RNA ligase is T4 RNA ligase.
20. The method of claim 17 , wherein said RNA ligase is T4 RNA ligase 1.
21. The method of claim 17 , wherein said RNA ligase is T4 RNA ligase 2.
22. The method of claim 17 , wherein said RNA ligase is T4 RNA ligase 2 truncated.
23. The method of claim 17 , wherein said RNA ligase is CircLigase RNA ligase.
24. The method of claim 1 , wherein said first nucleic acid form is DNA, said second nucleic acid form is RNA, said first ligase that has a preference for said first nucleic acid form is a DNA ligase, and said second ligase that has a preference for said second nucleic acid form is a RNA ligase.
25. The method of claim 24 , wherein said DNA is cell-free DNA.
26. The method of claim 25 , wherein said cell-free DNA is cell-free microbial DNA.
27. The method of claim 26 , wherein said DNA is bacterial cell-free DNA.
28. The method of claim 24 , wherein said RNA is microbial RNA.