Method for native strand sequencing of enriched genomic regions
AssigneeNanoHyb, LLC
InventorsJonathon Hill, Timothy Jenkins
Life of this patent
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Abstract
Provided herein, inter alia, are methods for sequencing a polynucleotide. In addition, provided herein are methods for detecting a base modification in polynucleotide, such as methylation calling, by sequencing only the native strand. The key innovation in this method is the creation of a second strand following hybridization that is configured to only allow adapter ligation to one end. This limits sequencing to the native strand, which is the only strand with potential modifications.
Claims36
A method for detecting a base modification in a target polynucleotide, the method comprising:
- (a) contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the target polynucleotide to create an adapter-target polynucleotide, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the adapter-target polynucleotide with one or more tags under conditions promoting hybridization of the one or more tags to the adapter-target polynucleotide to create a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide;
- (c) contacting the tagged adapter-target polynucleotide with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a pulldown complex;
- (d) isolating the pulldown complex;
- (e) optionally eluting the adapter-target polynucleotide from the pulldown complex;
- (f) contacting the pulldown complex or eluted adapter-target polynucleotide with a first polymerase under conditions promoting creation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide comprises a 3′ protective group or a 5′ protective group;
- (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions promoting ligation of the sequencing adapter to the adapter-target polynucleotide to create a sequencing complex;
- and
- (h) sequencing the sequencing complex, wherein the sequencing comprises sequencing of the target polynucleotide sequence without sequencing the complementary adapter-target polynucleotide,
- thereby detecting the base modification in the target polynucleotide.
A method for sequencing a target polynucleotide, the method comprising:
- (a) contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the target polynucleotide to create an adapter-target polynucleotide, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the adapter-target polynucleotide with one or more tags under conditions promoting hybridization of the one or more tags to the adapter-target polynucleotide to create a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide;
- (c) contacting the tagged adapter-target polynucleotide with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a pulldown complex;
- (d) isolating the pulldown complex;
- (e) optionally eluting the adapter-target polynucleotide from the pulldown complex;
- (f) contacting the pulldown complex or eluted adapter-target polynucleotide with a first polymerase under conditions promoting creation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide comprises a 3′ protective group or a 5′ protective group;
- (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions promoting ligation of the sequencing adapter to the adapter-target polynucleotide to create a sequencing complex;
- and
- (h) sequencing the sequencing complex, wherein the sequencing comprises sequencing of the target polynucleotide sequence without sequencing the complementary adapter-target polynucleotide,
- thereby sequencing the target polynucleotide, optionally wherein the target polynucleotide comprises one or more base modifications.
A method for quantifying a base modification in a target polynucleotide, the method comprising:
- (a) contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the target polynucleotide to create an adapter-target polynucleotide, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the adapter-target polynucleotide with one or more tags under conditions promoting hybridization of the one or more tags to the adapter-target polynucleotide or the adapter-target polynucleotide to the one or more tags to create a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide;
- (c) contacting the tagged adapter-target polynucleotide with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a pulldown complex;
- (d) isolating the pulldown complex;
- (e) optionally eluting the adapter-target polynucleotide from the pulldown complex;
- (f) contacting the pulldown complex or eluted adapter-target polynucleotide with a first polymerase under conditions promoting creation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide comprises a 3′ protective group or a 5′ protective group;
- (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions promoting ligation of the sequencing adapter to the adapter-target polynucleotide to create a sequencing complex;
- and
- (h) sequencing the sequencing complex, wherein the sequencing comprises sequencing of the target polynucleotide sequence without sequencing the complementary adapter-target polynucleotide,
- thereby quantifying the base modification in the target polynucleotide.
A method for detecting a plurality of base modifications in a plurality of target polynucleotides, the method comprising:
- (a) contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the plurality of target polynucleotides to create a plurality of adapter-target polynucleotides, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the plurality of adapter-target polynucleotides with one or more tags under conditions promoting hybridization of the one or more tags to the plurality of adapter-target polynucleotides to create a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides;
- (c) contacting the plurality of tagged adapter-target polynucleotides with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a plurality of pulldown complexes;
- (d) isolating the plurality of pulldown complexes;
- (e) optionally eluting the plurality of adapter-target polynucleotides from the plurality of pulldown complexes;
- (f) contacting the plurality of pulldown complexes or plurality of eluted adapter-target polynucleotides with a first polymerase under conditions promoting creation of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides comprise a 3′ protective group or a 5′ protective group;
- (g) contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions promoting ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to create a plurality of sequencing complexes;
- and
- (h) sequencing the plurality of sequencing complexes, wherein the sequencing comprises sequencing of the plurality of target polynucleotide sequences without sequencing the plurality of complementary adapter-target polynucleotides,
- thereby detecting a plurality of base modifications in the plurality of target polynucleotides.
A method for sequencing a plurality of target polynucleotides, the method comprising:
- (a) contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the plurality of target polynucleotides to create a plurality of adapter-target polynucleotides, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the plurality of adapter-target polynucleotides with one or more tags under conditions promoting hybridization of the one or more tags to the plurality of adapter-target polynucleotides to create a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides;
- (c) contacting the plurality of tagged adapter-target polynucleotides with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a plurality of pulldown complexes;
- (d) isolating the plurality of pulldown complexes;
- (e) optionally eluting the plurality of adapter-target polynucleotides from the plurality of pulldown complexes;
- (f) contacting the plurality of pulldown complexes or plurality of eluted adapter-target polynucleotides with a first polymerase under conditions promoting creation of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides comprise a 3′ protective group or a 5′ protective group;
- (g) contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions promoting ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to create a plurality of sequencing complexes;
- and
- (h) sequencing the plurality of sequencing complexes, wherein the sequencing comprises sequencing of the plurality of target polynucleotide sequences without sequencing the plurality of complementary adapter-target polynucleotides,
- thereby sequencing the plurality of target polynucleotides, optionally wherein the plurality of target polynucleotides comprise one or more base modifications.
A method for detecting a plurality of base modifications in a target polynucleotide, the method comprising:
- (a) contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the target polynucleotide to create an adapter-target polynucleotide, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the adapter-target polynucleotide with one or more tags under conditions promoting hybridization of the one or more tags to the adapter-target polynucleotide to create a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide;
- (c) contacting the tagged adapter-target polynucleotide with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a pulldown complex;
- (d) isolating the pulldown complex;
- (e) optionally eluting the adapter-target polynucleotide from the pulldown complex;
- (f) contacting the pulldown complex or eluted adapter-target polynucleotide with a first polymerase under conditions promoting creation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide comprises a 3′ protective group or a 5′ protective group;
- (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions promoting ligation of the sequencing adapter to the adapter-target polynucleotide to create a sequencing complex;
- and
- (h) sequencing the sequencing complex, wherein the sequencing comprises sequencing of the target polynucleotide sequence without sequencing the complementary adapter-target polynucleotide,
- thereby detecting the plurality of base modifications in the target polynucleotide.
A method for detecting a base modification in a plurality of target polynucleotides, the method comprising:
- (a) contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions promoting ligation of the one or more adapters to the plurality of target polynucleotides to create a plurality of adapter-target polynucleotides, wherein the one or more adapters comprise a primer binding site;
- (b) contacting the plurality of adapter-target polynucleotides with one or more tags under conditions promoting hybridization of the one or more tags to the plurality of adapter-target polynucleotides to create a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides;
- (c) contacting the plurality of tagged adapter-target polynucleotides with a solid support, under conditions promoting hybridization of the one or more tags to the solid support to create a plurality of pulldown complexes;
- (d) isolating the plurality of pulldown complexes;
- (e) optionally eluting the plurality of adapter-target polynucleotides from the plurality of pulldown complexes;
- (f) contacting the plurality of pulldown complexes or plurality of eluted adapter-target polynucleotides with a first polymerase under conditions promoting creation of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides comprise a 3′ protective group or a 5′ protective group;
- (g) contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions promoting ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to create a plurality of sequencing complexes;
- and
- (h) sequencing the plurality of sequencing complexes, wherein the sequencing comprises sequencing of the plurality of target polynucleotide sequences without sequencing the plurality of complementary adapter-target polynucleotides,
- thereby detecting a base modifications in the plurality of target polynucleotides.
The method of any one of the above claims, further comprising:
- (i) analyzing the sequenced sequencing complex to detect the base modification.
The method of any one of the above claims, wherein the sequencing comprises identifying a base modification in the target polynucleotide.
The method of any one of the above claims, further comprising repeating steps (a) through (i) at least once with one or more additional adapters.
The method of any one of the above claims, wherein the target polynucleotide is isolated from a biological sample prior to step (a).
The method of any one of the above claims, wherein the target polynucleotide comprises genomic DNA (gDNA) or cell-free DNA (cfDNA).
The method of any one of the above claims, wherein the target polynucleotide is fragmented prior to step (a).
The method of any one of the above claims, wherein the one or more adapters further comprise a first barcode.
The method of any one of the above claims, wherein the one or more tags comprise streptavidin, biotin, maltose, maltose binding protein, glutathione, glutathione S-transferase, chitin, chitin binding protein, an aptamer, an antigen, SpyCatcher, SpyTag, or an antibody.
The method of any one of the above claims, wherein the one or more tags target the same region of the target polynucleotide.
The method of any one of the above claims, wherein the solid support comprises streptavidin, biotin, maltose, maltose binding protein, glutathione, glutathione S-transferase, chitin, chitin binding protein, an aptamer, an antigen, SpyCatcher, SpyTag, or an antibody.
The method of any one of the above claims, wherein the solid support is a bead or a column.
The method of any one of the above claims, wherein the elution of step (e) comprises NaOH, heat, a strand-displacing polymerase, or a polymerase with exonuclease activity.
The method of any one of the above claims, wherein the 3′ protective group or the 5′ protective group prevents sequencing of the complementary adapter-target polynucleotide.
The method of any one of the above claims, wherein the 3′ protective group comprises a nucleotide overhang.
The method of any one of the above claims, the primer does not comprise a 5′ phosphate group.
The method of any one of the above claims, the sequencing adapter comprises a second barcode.
The method of any one of the above claims, wherein the base modification comprises a 5-methylcytosine (5mC), a 5-hydroxymethylcytosine (5hmC), a N 6-methyladenine (6 mA), a 7-methylguanine (m7G), 2′-O-methylation (2′-O-Methyl), or a bromodeoxyuridine (BrdU).
The method of any one of the above claims, wherein the method does not comprise bisulfite conversion or amplification of the target polynucleotide.
The method of any one of the above claims, wherein the method does not comprise reduced representation bisulfite sequencing (RRBS) or reduced representation methylation sequencing (RRMS).
The method of any one of the above claims, wherein the first ligase or the second ligase is a T4 DNA ligase, a Quick ligase, a T3 ligase, a T7 ligase, or a Tag ligase.
The method of any one of the above claims, wherein the first polymerase is a Taq polymerase, a BST polymerase, a sulfolobus polymerase, a therminator polymerase, a Klenow polymerase, a deep vent polymerase.
The method of any one of the above claims, wherein sequencing is performed on a sequencer capable of identifying modified bases.
The method of any one of the above claims, wherein the base modification in the target polynucleotide is retained until sequencing.
The method of any one of the above claims, wherein the method enriches the base modification in the target polynucleotide between about 300-fold to about 2400-fold compared to whole genome sequencing.
Dependencies are inferred from the claim text.