cDNA library for nucleic acid sequencing
The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.
1. A method for preparing a library of DNA copies of a plurality of RNA molecules, comprising:
(a) fragmenting the plurality of RNA molecules to produce RNA fragments having a 5′ end and a 3′ end;
(b) producing RNA constructs by adding a 5′-RNA oligonucleotide adaptor segment so that it becomes ligated to the 5′ end of the RNA fragments and adding a 3′-RNA oligonucleotide adaptor segment so that it becomes ligated to the 3′ end of the RNA fragments;
(c) preparing double-stranded DNA copies of the RNA constructs; and
(d) producing a library of circularized double-stranded DNA copies of said RNA fragments, wherein the circularized DNA copies comprise an adaptor that contains a complete DNA copy of the entire 5′-RNA oligonucleotide adaptor segment joined to the entire 3′-RNA oligonucleotide adaptor segment.
2. The method of claim 1 , wherein step (a) comprises fragmenting said plurality of RNA molecules to produce fragments that are between about 250 nucleotides and about 450 nucleotides long.
3. The method of claim 1 , wherein said plurality of RNA molecules together represents a full transcriptome.
4. The method of claim 1 , wherein the library comprises at least 90 percent of a transcriptome sequence.
5. The method of claim 1 , wherein the fragments produced in step (a) are between about 250 nucleotides and about 450 nucleotides long.
6. The method of claim 1 , further comprising inserting one or more additional adaptors into the circular double-stranded DNA molecules,
wherein each adaptor is inserted by a process that comprises cleaving the circular DNA to produce linear DNA, introducing adaptor segments onto one or both ends of the linear DNA, and then recircularizing the linear DNA to produce circular double-stranded DNA containing the inserted adaptor.
7. A method of sequencing a plurality of RNA molecules, comprising:
(i) preparing a library of circular double-stranded DNA copies of the RNA molecules according to the method of claim 1 ; and then
(ii) sequencing double-stranded DNA copies in the library, thereby obtaining sequences of the RNA molecules.
8. The method of claim 7 , wherein the sequencing comprises hybridizing a nucleic acid probe to the adaptor.