Patent Document ID: 9920367
Application ID: 14105704
Patent Flag: 1

Claim One:
1. A method of amplifying and sequencing low abundance transcripts, comprising: (a) determining a list of potential primer binding sites in a mammalian transcriptome that have an open configuration and are positioned adjacent to a unique region in the mammalian transcriptome; (b) determining at least one set of gene pool specific heptamer primers based on the list of potential primer binding sites using an iterative randomized algorithm, wherein each of the gene pool specific heptamer primers complementary binds to at least one of the potential primer binding sites and is capable of generating an amplicon which has: (i) a length between 50 and 300 bp; (ii) a GC content not exceeding 58%; (iii) at least one primer-binding site having a ΔG≥−2 Kcal/mol; (iv) forward and reverse primer-binding sites in an open configuration; and (v) at least one uniquely mappable 32 mer end; (c) amplifying at least a portion of the mammalian transcriptome using the at least one set of gene pool specific heptamer primers to form amplicons; (d) ligating adapter oligos to the amplicons to form adapted amplicons; (e) amplifying the adapted amplicons; and (f) subjecting the amplified, adapted amplicons to synthesis-based sequencing.