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US 2023/0144391 A1Published application
Next Generation Sequencing
AssigneeMorava, Inc.
InventorMike Mkhitar MORADIAN
Life of this patent
Estimating the term…
Abstract
An improved method for Next Generation Sequencing which relies on the presence of the same distinct unique molecular identifier (UMI) located at each end of a linear nucleic acid molecule so that sequence reads of approximately 2 kb or longer are obtained, and which allows generation of a genomic map without the need of a reference sequence.
Claims19
A method for generating a next generation sequencing library of any nucleic acid product comprising
- (a) isolating genomic DNA (gDNA), cDNA, or RNA from an organism;
- (b) fragmenting the gDNA, cDNA, or RNA;
- (c) isolating the gDNA, cDNA, or RNA fragments of step (b) and optionally conducting end-repair and/or A-tailing on each gDNA, cDNA, or RNA fragment;
- (d) obtaining a collection of arbitrary and unique molecular identifier (UMI) adapters, each adapter comprising a different UMI flanked by at least one deoxyuracil (dU) with the form of dU/UMI/dU;
- (e) attaching a different dU/UMI/dU adapter to each end of the gDNA, cDNA, or RNA fragment of step (c) to form a collection, wherein both ends of each gDNA, cDNA, or RNA fragment contains a dU/UMI/dU adapter to form dU/UMI/dU/gDNA/dU/UMI/dU fragments, dU/UMI/dU/cDNA/dU/UMI/dU fragments, or dU/UMI/dU/RNA/dU/UMI/dU fragments and wherein each end of the fragment of step (c) contains a different dU/UMI/dU adapter;
- (f) fragmenting the collection of dU/UMI/dU/gDNA/dU/UMI/dU fragments, dU/UMI/dU/cDNA/dU/UMI/dU fragments, or dU/UMI/dU/RNA/dU/UMI/dU fragments into smaller fragments to form dU/UMI/dU/gDNA and gDNA/dU/UM/dU fragments, dU/UMI/dU/cDNA and cDNA/dU/UM/dU fragments, or dU/UMI/dU/RNA and RNA/dU/UM/dU fragments;
- (g) optionally isolating the dU/UMI/dU/gDNA and gDNA/dU/UM/dU fragments, dU/UMI/dU/cDNA and cDNA/dU/UM/dU fragments, or dU/UMI/dU/RNA and RNA/dU/UM/dU fragments;
- (h) self-ligating each of the fragments of step (e) to form circular molecules;
- (i) contacting the self-ligated circular molecules of step (g) with a USER enzyme to create a linear gDNA, cDNA, or RNA, wherein each end of the USER treated gDNA, cDNA, or RNA has the same UMI at each end;
- (j) conducting end-repair on the USER treated linear gDNA, cDNA, or RNA of step (h);
- (k) ligating sequencing adapters comprising sequencing primers and index sequences to each end of the linear gDNA, cDNA, or RNA of step (i);
- (l) preparing a collection of primer extensions comprising a primer and a sequencing primer tail;
- (m) performing PCR extension using random primers with sequencing adapter tails on each gDNA, cDNA, or RNA of step (j) using the primer extensions of step (k);
- (n) generating a population of PCR fragments with different lengths, wherein each PCR fragment contains a UMI sequence;
- and
- (o) isolating the PCR products of step (m) for sequencing.
13 more elements
The method according to, wherein the dU/UMI/dU/gDNA and gDNA/dU/UM/dU fragments, dU/UMI/dU/cDNA and cDNA/dU/UM/dU fragments, or dU/UMI/dU/RNA and RNA/dU/UM/dU fragments of step (f) are 2-4 kb in length.
A method for generating a next generation sequencing library of any nucleic acid product comprising
- (a) isolating genomic DNA (gDNA), cDNA, or RNA from an organism;
- (b) fragmenting the gDNA, cDNA, or RNA;
- (c) isolating the gDNA, cDNA, or RNA fragments of step (b) and optionally conducting end-repair and/or A-tailing on each gDNA, cDNA, or RNA fragment;
- (d) obtaining a collection of arbitrary and unique molecular identifier (UMI) adapters, each adapter comprising a different UMI flanked by at least one deoxyuracil (dU) with the form of dU/UMI/dU;
- (e) attaching a different dU/UMI/dU adapter to each end of the gDNA, cDNA, or RNA fragment of step (c) to form a collection, wherein both ends of each gDNA, cDNA, or RNA fragment contains a dU/UMI/dU adapter to form dU/UMI/dU/gDNA/dU/UMI/dU fragments, dU/UMI/dU/cDNA/dU/UMI/dU fragments, or dU/UMI/dU/RNA/dU/UMI/dU fragments, wherein each end of the fragment of step (c) contains a different dU/UMI/dU adapter;
- (f) fragmenting the collection of dU/UMI/dU/gDNA/dU/UMI/dU fragments, dU/UMI/dU/cDNA/dU/UMI/dU fragments, or dU/UMI/dU/RNA/dU/UMI/dU fragments into smaller fragments to form dU/UMI/dU/gDNA and gDNA/dU/UM/dU fragments, dU/UMI/dU/cDNA and cDNA/dU/UM/dU fragments, or dU/UMI/dU/RNA and RNA/dU/UM/dU fragments;
- (g) optionally isolating the dU/UMI/dU/gDNA and gDNA/dU/UM/dU fragments, dU/UMI/dU/cDNA and cDNA/dU/UM/dU fragments, or dU/UMI/dU/RNA and RNA/dU/UM/dU fragments;
- (h) self-ligating each of the fragments of step (f) to form a collection of circular molecules;
- (i) fragmenting the collection of circular molecules of step (h) to create a collection of linear gDNA, cDNA, or RNA molecules, wherein the dU/UMI/dU is internal to the two ends of each linear molecule;
- (j) contacting the collection of linear molecules of step (i) with a USER enzyme to cleave the dU/UMI/dU sequence and to create two linear gDNA, cDNA, or RNA fragments from each linear molecule in the collection;
- (k) conducting end-repair on the USER treated linear gDNA, cDNA, or RNA fragment of step (j);
- (l) ligating sequencing adapters comprising sequencing primers and, optionally, index sequences to each end of the linear gDNA, cDNA, or RNA of step (k);
- (m) preparing a collection of primer extensions comprising a sequencing primer tail;
- (n) performing PCR extension on each gDNA, cDNA, or RNA of step (l) using the sequencing primers of step (m);
- (o) generating a population of PCR fragments with different lengths, wherein each PCR fragment contains a UMI sequence;
- and
- (p) isolating the PCR products of step (o) for sequencing.
14 more elements
A method of generating a next generation targeted sequencing library for a nucleic acid product of interest comprising
- (a) isolating genomic DNA (gDNA), cDNA, or RNA from an organism;
- (b) fragmenting the gDNA, cDNA, or RNA;
- (c) isolating the gDNA, cDNA, or RNA fragments of step (b);
- (d) performing PCR extension on the fragments of step (c) using a collection of forward primers and a collection of reverse primers, wherein the forward primer comprises a sequence specific to the nucleic acid product of interest joined to a random unique molecular identifier (UMI) joined to a sequencing adapter tail and the reverse primer comprising a sequence specific to the nucleic acid product of interest;
- (e) performing PCR primer extension using random primers joined to the forward primer sequencing adapter tail;
- and
- (f) isolating the primer extensions of step (e) for sequencing.
4 more elements
Dependencies are inferred from the claim text.