CPC C12Q 1/6874 (2013.01) [C12N 15/1068 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6876 (2013.01); C12Q 2600/156 (2013.01)] | 34 Claims |
1. A method comprising:
A) obtaining a plurality of cDNA sequences from a pool of poly-adenylated mRNA obtained from a biological sample of a subject, wherein
each respective cDNA sequence in the plurality of cDNA sequences is unfragmented,
the plurality of cDNA sequences comprises a first subset of cDNA sequences and a second subset of cDNA sequences,
each respective cDNA sequence in the first subset of cDNA sequences maps to a respective target gene in a plurality of target genes,
each respective cDNA sequence in the second subset of cDNA sequences maps to an off-target portion of a reference genome not represented by the plurality of target genes, and
each respective target gene in the plurality of target genes is characterized by a respective plurality of transcripts comprising overlapping isoforms in a plurality of isoforms for the respective target gene, wherein each respective transcript in the respective plurality of transcripts is a respective isoform for the respective target gene;
B) exposing the plurality of cDNA sequences to a plurality of nucleic acid baits each of length that is between K1 and K2 residues, thereby forming a plurality of nucleic acid bait-sequence read complexes, wherein:
K1 and K2 are positive integers,
the plurality of nucleic acid baits comprises at least 2×103 nucleic acid baits,
each respective nucleic acid bait in the plurality of nucleic acid baits that hybridizes to a cDNA sequence mapping to a respective target gene in the plurality of target genes:
(i) selectively hybridizes to a first subset of transcripts, in a plurality of subsets of transcripts in the respective plurality of transcripts for the respective target gene, or
(ii) selectively hybridizes to another subset of transcripts, other than the first subset of transcripts, in the plurality of subsets of transcripts in the respective plurality of transcripts for the respective target gene, and
for each respective target gene in the plurality of target genes, the plurality of nucleic acid baits hybridizes to all of the overlapping isoforms in the plurality of isoforms for the respective target gene;
C) selectively capturing the plurality of nucleic acid bait—sequence read complexes; and
D) analyzing the plurality of nucleic acid bait—sequence read complexes captured by the selectively capturing C).
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