CPC C12N 15/1093 (2013.01) [C12Q 1/6806 (2013.01)] | 13 Claims |
1. A method of generating a next generation UMI targeted sequencing library for contiguous sequence formation for a nucleic acid of interest comprising
(a) isolating genomic DNA (gDNA), cDNA, or RNA from an organism;
(b) optionally fragmenting the gDNA, cDNA, or RNA;
(c) optionally isolating the gDNA, cDNA, or RNA fragments of step (b);
(d) performing PCR extension on the genomic DNA (gDNA), cDNA, or RNA from step (a), (b), or (c) using deoxynucleotide triphosphates (dNTPs) only to obtain a collection of PCR products using
(i) a first collection of forward primers, wherein each forward primer comprises in 5′ to 3′ order a first sequencing adapter tail, a random unique molecular identifier (UMI), which will serve as a starting sequence point in a de novo assembly of the nucleic acid of interest, and a sequence specific to the nucleic acid of interest; and
(ii) a collection of reverse primers, wherein each reverse primer consists of a sequence specific to the nucleic acid of interest;
(e) performing PCR primer extension on the collection of PCR products of step (d) to obtain a collection of double stranded PCR primer extension products using
(i) a second collection of forward primers comprising in 5′ to 3′ order a second forward primer sequencing adapter tail and a random primer sequence; and
(ii) a reverse primer consisting of the first sequencing adapter tail as set forth in step (d) (i);
(f) isolating the collection of double stranded PCR primer extension products of step (e) for sequencing;
(g) sequencing the isolated collection of double stranded PCR primer extension products of step (f), wherein the sequencing results in a collection of sequences and wherein one of the sequences of the collection of sequences comprises the random UMI;
(h) identifying the sequence comprising the random UMI obtained from the sequencing of step (g); and
(i) overlapping each sequence of the collection of sequences obtained from step (g) to form a contiguous sequence, wherein the sequence which comprises the random UMI identified in step (h) is used as a starting point for forming the contiguous sequence.
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