US 12,129,464 B2
Next generation sequencing
Mike Mkhitar Moradian, Glendale, CA (US)
Assigned to MORAVA, INC., Glendale, CA (US)
Filed by MORAVA INC., Glendale, CA (US)
Filed on Dec. 5, 2022, as Appl. No. 18/061,825.
Application 18/061,825 is a continuation of application No. PCT/US2021/055477, filed on Oct. 18, 2021.
Claims priority of provisional application 63/093,602, filed on Oct. 19, 2020.
Prior Publication US 2023/0144391 A1, May 11, 2023
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6806 (2018.01)
CPC C12N 15/1093 (2013.01) [C12Q 1/6806 (2013.01)] 13 Claims
OG exemplary drawing
 
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.