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 performing a primer extension reaction on RNA and DNA, comprising:
a) providing a sample comprising a mixture of single-stranded DNA and single-stranded RNA;
b) attaching a first adapter to said single-stranded DNA,
c) attaching a second adapter to said single-stranded RNA;
d) annealing a first primer to said first adapter and annealing a second primer to said second adapter;
e) extending said annealed first primer on said single-stranded DNA to form double-stranded DNA; and
f) extending said annealed second primer on said single-stranded RNA to form a double-stranded DNA-RNA hybrid.
2. The method of claim 1 , wherein said attaching said first adapter comprises ligating said first adapter to a 3′ end of said single-stranded DNA.
3. The method of claim 2 , wherein said ligating said first adapter is performed by a ligase selected from the group consisting of CircLigase II, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated, and any combination thereof.
4. The method of claim 1 , wherein said attaching said second adapter comprises ligating said second adapter to a 3′ end of said single-stranded RNA.
5. The method of claim 4 , wherein said ligating said second adapter is performed using an RNA ligase.
6. The method of claim 4 , wherein said ligating said second adapter is performed using T4 RNA ligase 2 or T4 DNA ligase.
7. The method of claim 1 , wherein said single-stranded DNA is cell-free DNA.
8. The method of claim 1 , wherein said sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.
9. The method of claim 1 , wherein said extending said annealed first primer on said single-stranded DNA is performed by a DNA polymerase.
10. The method of claim 1 , wherein said extending said annealed first primer on said single-stranded DNA is performed by Bst 2.0 DNA polymerase.
11. The method of claim 1 , wherein said extending said annealed second primer on said single-stranded RNA is performed by a reverse transcriptase.
12. The method of claim 1 , further comprising adding at least one non-templated nucleotide to a first primer extension strand.
13. The method of claim 12 , wherein said at least one non-templated nucleotide is a deoxycytidine.
14. The method of claim 12 , wherein said at least one non-templated nucleotide is added to a 3′ end.
15. The method of claim 12 , wherein said at least one non-templated nucleotide is up to eight nucleotides.
16. The method of claim 12 , wherein said at least one non-templated nucleotide is three, four, or five non-templated nucleotides.
17. The method of claim 12 , wherein said at least one non-templated nucleotide is one non-templated nucleotide.
18. The method of claim 12 , wherein said at least one non-templated nucleotide forms a first overhang.
19. The method of claim 18 , further comprising adding at least one second non-templated nucleotide to a second primer extension strand forming a second overhang.
20. The method of claim 19 , further comprising hybridizing a third adapter to said first overhang and a fourth adapter to said second overhang.
21. The method of claim 20 , further comprising sequencing said third adapter and said fourth adapter and sequences attached to said third adapter and said fourth adapter.
22. The method of claim 20 , further comprising (i) identifying sequences associated with said third adapter as originating from said DNA in said mixture of single-stranded DNA and single-stranded RNA and (ii) identifying sequences associated with said fourth adapter as originating from said RNA in said mixture of single-stranded DNA and single-stranded RNA.