METHODS FOR TRANSCRIPT ANALYSIS
The invention takes a unique approach to transcript analysis that provides a novel DGE technology based on single-molecule sequencing. More particularly, the invention relates to methods and compositions for analyzing and identifying genes and gene expression and transcript profiles using a DGE-based technology and single molecule sequencing that does not require amplification or fragmentation.
1 . A method for analyzing RNA transcripts, the method comprising sequencing a first strand cDNA via single-molecule sequencing thereby obtaining transcript information, wherein the method does not comprise a step of RNA or cDNA amplification.
2 . The method of claim 1 , wherein the method does not comprise RNA or cDNA fragmentation.
3 . The method of claim 1 , wherein the method comprising the steps of:
copying a RNA to form a cDNA;
polyadenylating the cDNA;
hybridizing the polyadenylated cDNA to a primer bound to a surface; and
conducting single-molecule sequencing of the cDNA.
4 . The method of claim 3 , further comprising obtaining a RNA sample from a tissue or body fluid of a subject.
5 . The method of claim 4 , wherein the subject is a human.
6 . The method of claim 1 , wherein the RNA transcripts are from a human gene.
7 . The method of claim 3 , wherein the surface comprises a glass surface.
8 . The method of any of claims 1 to 7 , wherein the RNA or cDNA is from about 20 nt to about 500K nt.
9 . The method of claim 8 , wherein the RNA or cDNA is from about 100 nt to about 100K nt.
10 . A method for detecting a sequence in a sample that does not align with a reference sequence thought to be in the sample, the method comprising the steps of:
copying said RNA to form a cDNA;
polyadenylating the cDNA;
hybridizing the polyadenylated cDNA to a primer bound to a surface;
conducting sequencing by synthesis;
aligning the RNA to a reference sequence;
collecting RNA that does not align to said reference sequence; and
determining the origin of the unaligned RNA.
11 . The method of claim 10 , further comprising obtaining a RNA sample from a tissue or body fluid of a subject.
12 . The method of claim 11 , wherein the subject is a human.
13 . The method of claim 10 , wherein the RNA transcripts are from a human gene.
14 . The method of claim 13 , wherein the surface comprises a glass surface.
15 . The method of any of claim 10 to claim 14 , wherein the RNA or cDNA is from about 20 nt to about 500K nt.
16 . The method of claim 15 , wherein the RNA or cDNA is from about 100 nt to about 100K nt.