Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing

Abstract

Provided herein aremethods, compositions and kits for the generation of bisulfite-converted next generation sequencing (NGS) libraries. The methods, compositions and kits provided herein can be useful, for example, for the production of libraries from genomic DNA that allow for determination of the methylation status across the genome, i.e. the methylome. The methods, compositions and kits provided herein can also be utilized to query methylation status at a particular genomic locus or loci. Moreover, the methods provided herein can be employed for high-throughput sequencing of bisulfite-converted DNA while maintaining the directional (strandedness) information of the original nucleic acid sample.

1 . A method for generating bisulfite-converted directional nucleic acid libraries, the method comprising:
 a. fragmenting double-stranded DNA, thereby generating double-stranded DNA fragments;   b. performing end repair on the DNA fragments;   c. ligating a single oligonucleotide adapter duplex, wherein one strand of the adapter is capable of ligation to a 5′ end of the DNA fragment and the other stand is incapable of ligation at a 3′ end of the DNA fragment, to both ends of each DNA fragment; or, alternatively, ligating distinct oligonucleotide adapters of similar construct to both ends of each DNA fragment, wherein at least one cytosine residue in the ligation compatible arm of the adapter(s) has been replaced with a cytosine analog resistant to bisulfite treatment;   d. extending the 3′ end of the DNA fragments with a DNA polymerase;   e. denaturing DNA, thereby creating single-stranded DNA fragments;   f. subjecting the single-stranded DNA fragments to bisulfite treatment, thereby converting cytosine residues to uracils and creating unique PCR priming sites at the 5′ and 3′ ends of the DNA fragments; and   g. performing PCR with oligonucleotide primers corresponding to the unique priming sites.   
     
     
         2 . The method of  claim 1 , further comprising an additional step of sequencing the amplified products. 
     
     
         3 . The method of  claim 1 , wherein the double-stranded DNA comprises genomic DNA. 
     
     
         4 . The method of  claim 1 , wherein the 5′ and/or 3′ ends of the oligonucelotide of the duplex-forming adapter(s) incapable of ligation are blocked and enzymatically unreactive to prevent adapter dimer formation. 
     
     
         5 . The method of  claim 1 , wherein the 3′ end of the oligonucleotide of the duplex-forming adapter incapable of ligation is blocked with a terminal dideoxycytosine. 
     
     
         6 . The method of  claim 1 , wherein the 5′ end of the oligonucleotide of the duplex-forming adapter incapable of ligation contains a biotin moiety. 
     
     
         7 . The method of  claim 1 , wherein step g) comprises annealing to the DNA fragments a sequence-specific oligonucleotide primer, or multiple sequence-specific oligonucleotide primers, further comprising an additional barcode sequence. 
     
     
         8 . The method of  claim 1 , wherein the cytosine analog resistant to bisulfite treatment is 5-methylcytosine. 
     
     
         9 . The method of  claim 1 , wherein the cytosine analog resistant to bisulfite treatment is 5-hydroxymethylcytosine. 
     
     
         10 . The method of  claim 1 , wherein the cytosine analog resistant to bisulfite treatment is 5-propynylcytosine. 
     
     
         11 . The method of  claim 1 , wherein 5-methylcytosine capture is performed prior to step f) and wherein the cytosine analog resistant to bisulfite treatment is a cytosine analog other than 5-methylcytosine. 
     
     
         12 . The method of  claim 11 , wherein 5-methylcytosine capture is performed using a methylcytosine binding protein. 
     
     
         13 . The method of  claim 11 , wherein 5-methylcytosine capture is performed using an anti-5-methylcytosine antibody. 
     
     
         14 . A method for generating bisulfite-converted directional nucleic acid libraries, the method comprising:
 a. fragmenting double-stranded DNA, thereby generating double-stranded DNA fragments;   b. performing end repair on the DNA fragments;   c. ligating a single oligonucleotide adapter forming a partial duplex to both ends of each DNA fragment; or alternatively, ligating distinct oligonucleotide adapters, each forming a partial duplex, to both ends of each DNA fragment, wherein at least one cytosine residue in one arm of the partial duplex adapter(s) has been replaced with a cytosine analog resistant to bisulfite treatment;   d. extending the ligated oligonucleotide adapter(s) with a DNA polymerase;   e. denaturing DNA, thereby creating single-stranded DNA fragments;   f. subjecting the single-stranded DNA fragments to bisulfite treatment, thereby converting cytosine residues to uracils and creating unique PCR priming sites at the 5′ and 3′ ends of the DNA fragments; and   g. performing PCR with oligonucleotide primers corresponding to the unique priming sites.   
     
     
         15 . The method of  claim 14 , further comprising an additional step of sequencing the amplified products. 
     
     
         16 . The method of  claim 14 , wherein the double-stranded DNA comprises genomic DNA. 
     
     
         17 . The method of  claim 14 , wherein step g) comprises annealing to the DNA fragments a sequence-specific oligonucleotide primer, or multiple sequence-specific oligonucleotide primers, further comprising an additional barcode sequence. 
     
     
         18 . The method of  claim 14 , wherein the cytosine analog resistant to bisulfite treatment is 5-methylcytosine. 
     
     
         19 . The method of  claim 14 , wherein the cytosine analog resistant to bisulfite treatment is 5-hydroxymethylcytosine. 
     
     
         20 . The method of  claim 14 , wherein the cytosine analog resistant to bisulfite treatment is 5-propynylcytosine. 
     
     
         21 . The method of  claim 14 , wherein 5-methylcytosine capture is performed prior to step f) and wherein the cytosine analog resistant to bisulfite treatment is a cytosine analog other than 5-methylcytosine. 
     
     
         22 . The method of  claim 21 , wherein 5-methylcytosine capture is performed using a methylcytosine binding protein. 
     
     
         23 . The method of  claim 21 , wherein 5-methylcytosine capture is performed using an anti-5-methylcytosine antibody. 
     
     
         24 - 36 . (canceled) 
     
     
         37 . A method for generating bisulfite-converted directional nucleic acid libraries, the method comprising:
 a. fragmenting double-stranded DNA, thereby generating double-stranded DNA fragments;   b. performing end repair on the DNA fragments;   c. ligating a single oligonucleotide adapter forming a partial duplex to both ends of each DNA fragment; or, alternatively, ligating distinct oligonucleotide adapters, each forming a partial duplex, to both ends of each DNA fragment;   d. extending the ligated oligonucleotide adapter(s) with a DNA polymerase, wherein the extension reaction is performed in presence of dATP, dGTP, dTTP and a dCTP analog resistant to bisulfite treatment;   e. denaturing DNA, thereby creating single-stranded DNA fragments;   f. subjecting the single-stranded DNA fragments to bisulfite treatment, thereby converting cytosine residues to uracils and creating unique PCR priming sites at the 5′ and 3′ ends of the DNA fragments; and   g. performing PCR with oligonucleotide primers corresponding to the unique priming sites.   
     
     
         38 . The method of  claim 37 , further comprising an additional step of sequencing the amplified products. 
     
     
         39 . The method of  claim 37 , wherein the double-stranded DNA comprises genomic DNA. 
     
     
         40 . The method of  claim 37 , wherein step g) comprises annealing to the DNA fragments a sequence-specific oligonucleotide primer, or multiple sequence-specific oligonucleotide primers, further comprising an additional barcode sequence. 
     
     
         41 . The method of  claim 37 , wherein the dCTP analog resistant to bisulfite treatment is 5-methyl dCTP. 
     
     
         42 . The method of  claim 37 , wherein the dCTP analog resistant to bisulfite treatment is 5-hydroxymethyl dCTP. 
     
     
         43 . The method of  claim 37 , wherein the dCTP analog resistant to bisulfite treatment is 5-propynyl dCTP. 
     
     
         44 . The method of  claim 37 , wherein 5-methylcytosine capture is performed prior to step f) and wherein dCTP analog resistant to bisulfite treatment is an analog other than 5-methyl dCTP. 
     
     
         45 . The method of  claim 44 , wherein 5-methylcytosine capture is performed using a methylcytosine binding protein. 
     
     
         46 . The method of  claim 44 , wherein 5-methylcytosine capture is performed using an anti-5-methylcytosine antibody. 
     
     
         47 . A method for querying the methylation status of a genomic DNA sample, the method comprising:
 a. fragmenting genomic DNA, thereby generating DNA fragments;   b. performing end repair on the DNA fragments;   c. ligating a single oligonucleotide adapter forming a partial duplex, wherein a long strand of the adapter is capable of ligation to a 5′ end of the DNA fragment and a shorter strand is incapable of ligation at a 3′ end of the DNA fragment, to both ends of each DNA fragment; or, alternatively, ligating distinct oligonucleotide adapters of similar construct, each forming a partial duplex, to both ends of each DNA fragment, wherein at least one cytosine residue in one arm of the partial duplex adapter(s) has been replaced with a cytosine analog resistant to bisulfite treatment;   d. extending the 3′ end of the DNA fragment with a DNA polymerase;   e. denaturing DNA, thereby creating single-stranded DNA fragments;   f. subjecting the single-stranded DNA fragments to bisulfite treatment, thereby converting cytosine residues to uracils and creating unique PCR priming sites at the 5′ and 3′ ends of the DNA fragments;   g. performing PCR with oligonucleotide primers corresponding to the unique priming sites; and   h. sequencing the amplified products.   
     
     
         48 . (canceled)