METHODS OF ANALYZING NUCLEIC ACIDS
Presented herein are methods and compositions for analyzing rare nucleic acid species. Some methods presented herein use DNA reassociation kinetics following thermal denaturation to define populations of nucleic acid sequences, e.g., highly abundant (e.g., cDNA from rRNA), moderately abundant, and less abundant or rare sequences (e.g., cDNA from mRNA).
1 . A method of analyzing rare nucleic acid species comprising:
(a) providing a library of double-stranded nucleic acids, wherein the library of double-stranded nucleic acids comprises complementary DNA (cDNA) prepared from RNA;
(b) denaturing the library;
(c) renaturing the library under conditions sufficient to renature abundant nucleic acid species, wherein a portion of the library comprising less abundant nucleic acid species does not renature;
(d) contacting the library with a nucleic acid binding protein that preferentially binds to double-stranded nucleic acids; and
(e) separating renatured abundant nucleic acid species from the less abundant nucleic acid species.
2 . The method of claim 1 , wherein separating comprises immobilizing the nucleic acid binding protein to a solid support.
3 . The method of claim 1 , wherein the nucleic acid binding protein comprises a transposase.
4 . The method of claim 3 , wherein the nucleic acid binding protein comprises Tn5 or TS-Tn5 transposase.
5 . The method of claim 1 , wherein the nucleic acid binding protein comprises adaptor sequences which comprise a binding moiety capable of binding to a solid support.
6 . The method of claim 1 , wherein the nucleic acid binding protein comprises adaptor sequences which comprise a binding moiety capable of binding to a solid support.
7 . The method of claim 6 , wherein the binding moiety comprises biotin and the solid support comprises streptavidin.
8 . The method of claim 1 , wherein the nucleic acid binding protein comprises adaptor sequences which are not compatible with downstream amplification and sequencing.
9 . The method of claim 1 , wherein denaturing comprises heat denaturing.
10 . The method of claim 9 , wherein heat denaturing comprises application of heat above 70° C., 75° C., 80° C., 85° C., 90° C., 91° C., 92° C., 93° C., 94° C., or above 95° C.
11 . The method of claim 1 , wherein renaturing comprises lowering the denaturation temperature to a temperature of about 40° C. to about 65° C. for a sufficient period of time to renature abundant nucleic acid species.
12 . The method of claim 11 , wherein the sufficient period of time comprises about 30 minutes to about 24 hours.
13 . The method of claim 1 , wherein denaturing comprises chemical denaturing.
14 . The method of claim 1 , further comprising sequencing the separated less abundant nucleic acid species.
15 .- 17 . (canceled)
18 . The method of claim 1 , wherein the nucleic acids in the library comprise adaptor sequences ligated to the ends of the nucleic acids.
19 . The method of claim 18 , wherein the adaptor sequences comprise one or more of: an amplification priming binding region, a sequencing primer binding region, and promotor sequences for in vitro transcription.
20 . The method of claim 1 , wherein the abundant nucleic acid species comprise highly repetitive sequences.
21 . The method of claim 1 , wherein the library comprises nucleic acid from a single cell or a small group of cells.
22 . The method of claim 1 , wherein the abundant nucleic acid species comprise host sequences and the less abundant nucleic acid species comprise pathogen content.
23 . The method of claim 1 , wherein the abundant nucleic acid species comprise duplicate libraries in a sequencing library.
24 . (canceled)