Method for preparing single-stranded DNA libraries

Abstract

This invention relates to methods of generating single stranded DNA libraries for use in amplification and sequencing reactions. In various aspects, the disclosed methods include: fragmenting DNA; polishing the fragments' ends; ligating the fragments to universal adaptors; performing strand displacement and extension of the nicked fragments; purifying the double-stranded ligation products; capturing the double-stranded ligation products onto a solid support; and isolating single stranded DNA library fragments, and binding these fragments to another solid support.

1 . A method for clonally isolating a library comprising a plurality of single stranded DNA molecules comprising:
 (a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules;   (b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to form a mixture of adaptor ligated DNA molecules;   (c) isolating a plurality of single stranded DNA molecules each comprising a first single stranded universal adaptor and a second single stranded universal adaptor to obtain a library; and   (d) delivering the single stranded DNA molecules into reactors such that a plurality of the reactors include one DNA molecule, thereby clonally isolating the library.   
     
     
         2 . A method for generating a library comprising a plurality of single stranded DNA molecules, comprising:
 (a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules;   (b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules, wherein the first universal adaptor contains a moiety that binds to a solid support;   (c) attaching to a solid support those DNA molecules comprising a first double stranded universal adaptor;   (d) removing adaptor ligated DNA molecules which have not attached to a solid support;   (e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules having a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and   (f) isolating a library of the released single stranded DNA molecules of step (e) away from those DNA molecules that remain attached to the solid support.   
     
     
         3 . The method according to  claim 1  or  2 , wherein the first universal double stranded adaptor or the second universal adaptor is attached by ligation. 
     
     
         4 . The method according to  claim 1 , wherein (f) is accomplished by: i) delivering the single stranded DNA molecules onto a location on a reactor array; or ii) delivering the single stranded DNA molecules into droplets in a water-in-oil emulsion. 
     
     
         5 . The method according to  claim 1  or  2 , further comprising the step of repairing single stranded nicks in the mixture of adaptor ligated DNA molecules using DNA repair and modifying enzymes. 
     
     
         6 . The method according to  claim 5 , wherein the DNA repair and modifying enzymes are selected from the group consisting of polymerase, ligase, kinase, and combinations thereof. 
     
     
         7 . The method according to  claim 6 , wherein the polymerase is  Bacillus stearothermophilus  polymerase 1, the ligase is T4 ligase, and the kinase is T4 polynucleotide kinase. 
     
     
         8 . The method according to  claim 1  or  2 , wherein the template DNA is selected from the group consisting of genomic DNA, cDNA, plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA. 
     
     
         9 . The method according to  claim 1  or  2 , wherein the template DNA comprises reverse transcripts. 
     
     
         10 . The method according to  claim 1  or  2 , wherein the fragmenting is performed by a means selected from the group consisting of enzymatic, chemical, and mechanical means. 
     
     
         11 . The method according to  claim 10 , wherein the enzymatic means is DNase 1. 
     
     
         12 . The method according to  claim 10 , wherein the mechanical means is nebulization. 
     
     
         13 . The method according to  claim 11 , wherein the DNase I digestion is performed at a temperature of 10-37° C. for 1-2 minutes. 
     
     
         14 . The method according to  claim 10 , wherein the enzymatic means is a restriction endonuclease. 
     
     
         15 . The method according to  claim 10 , wherein the mechanical means is selected from the group consisting of a French Press, a sonicator, a HydroShear, and a nebulizer. 
     
     
         16 . The method according to  claim 1  or  2 , wherein the fragmented DNA molecules are 50 bp to 700 bp in length. 
     
     
         17 . The method according to  claim 1  or  2 , wherein the compatible ends are blunt ends. 
     
     
         18 . The method according to  claim 17  wherein the blunt ends are created with an enzyme selected from the group consisting of Pfu polymerase, T4 DNA polymerase and Klenow fragments. 
     
     
         19 . The method according to  claim 1  or  2 , wherein the compatible ends include an A or T overhang. 
     
     
         20 . The method according to  claim 1  or  2 , wherein the first or second double stranded universal adaptor comprises phosphorothioate linkages. 
     
     
         21 . The method according to  claim 1  or  2 , wherein a biotin moiety is attached to the first or second double stranded universal adaptor. 
     
     
         22 . The method according to  claim 1  or  2 , wherein the first or second double stranded universal adaptor is ligated with T4 DNA ligase. 
     
     
         23 . The method according to  claim 1  or  2 , wherein either the first double stranded universal adaptors or the second double stranded universal adaptors or both double stranded universal adaptors comprise a discriminating key sequence. 
     
     
         24 . The method according to  claim 23 , wherein the discriminating key sequence is 3-12 nucleotides in length. 
     
     
         25 . The method according to  claim 23 , wherein the discriminating key sequence comprises at least one nucleotide selected from the group consisting of A, G, C, U, and T. 
     
     
         26 . The method according to  claim 1  or  2 , wherein first and second double stranded universal adaptor comprise a PCR priming sequence and a sequencing primer sequence. 
     
     
         27 . The method according to  claim 26 , wherein the PCR priming sequence is 10-20 base pairs in length. 
     
     
         28 . The method according to  claim 26  wherein the sequencing primer sequence is 10-20 base pairs in length. 
     
     
         29 . The method according to  claim 26 , wherein the PCR priming sequence and the sequencing primer sequence overlap. 
     
     
         30 . The method according to  claim 1  or  2 , wherein the mixture of adaptor ligated DNA molecules is separated by a method selected from the group consisting of gel electrophoresis, filtration, size exclusion chromatography, and sucrose sedimentation. 
     
     
         31 . The method according to  claim 1  or  2 , wherein the plurality of single stranded DNA molecules is attached to a DNA capture bead. 
     
     
         32 . The method according to  claim 31 , wherein the DNA capture bead comprises a component of a binding pair. 
     
     
         33 . The method according to  claim 31  wherein the binding pair is selected from the group consisting of avidin/biotin, ligand/receptor, antigen/antibody and complementary nucleotides. 
     
     
         34 . The method according to  claim 31 , wherein the DNA capture bead is a paramagnetic bead. 
     
     
         35 . The method according to  claim 1  or  2 , wherein the plurality of single stranded DNA molecules is obtained by a treatment selected from the group consisting of low salt treatment, high pH treatment, and chemical denaturation treatment. 
     
     
         36 . A method for generating a single stranded DNA library attached to solid supports comprising:
 (a) generating a plurality of single stranded DNA templates;   (b) attaching each of the plurality of ssDNA templates to a solid support; and   (c) isolating the solid supports on which the single stranded DNA templates are attached.   
     
     
         37 . A method for generating a single stranded DNA library attached to solid supports comprising:
 (a) fragmenting large template DNA molecules to generate a plurality of fragmented DNA molecules;   (b) attaching a first or second universal double stranded adaptor to a first end of each fragmented DNA molecule and a first or second universal adaptor to a second end of each fragmented DNA molecule to make a mixture of adaptor ligated DNA molecules;   (c) isolating those single stranded DNA molecules which comprise a first single stranded universal adaptor and a second single stranded universal adaptor; and   (d) attaching the isolated single stranded molecules from (c) to a solid support.   
     
     
         38 . The method according to  claim 37 , wherein the solid support is a DNA capture bead. 
     
     
         39 . The method according to  claim 37 , wherein the DNA is selected from the group consisting of genomic DNA, cDNA, plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA. 
     
     
         40 . The method according to  claim 37 , wherein the DNA is attached to the solid support via a binding pair. 
     
     
         41 . The method according to  claim 40 , wherein the binding pair is selected from the group consisting of avidin/biotin, ligand/receptor, antigen/antibody and complementary nucleotides. 
     
     
         42 . A library of mobile solid supports made by the method of  claim 37 . 
     
     
         43 . A nucleic acid molecule comprising a first adaptor, a fragment of template DNA, and a second adaptor, wherein the first adaptor and second adaptor each comprise a sequencing primer, a PCR primer, and a discriminating key sequence, and wherein the first adaptor and second adaptor, when dissociated, do not cross-hybridize to each other under stringent hybridization conditions. 
     
     
         44 . The nucleic acid molecule of  claim 43 , wherein the PCR primer is 10-20 base pairs in length. 
     
     
         45 . The nucleic acid molecule of  claim 43 , wherein the sequencing primer is 10-20 base pairs in length. 
     
     
         46 . The nucleic acid molecule of  claim 43 , wherein the discriminating key sequence is 3 to 12 base pairs in length. 
     
     
         47 . The nucleic acid molecule of  claim 43 , wherein the template DNA is selected from the group consisting of genomic DNA, cDNA plasmid DNA, cosmid DNA, artificial chromosome DNA, synthetic DNA, phasemid DNA, and phagemid DNA. 
     
     
         48 . The nucleic acid molecule of  claim 43 , wherein the nucleic acid molecule, when dissociated, has minimal cross-hybridization to dissociated template DNA. 
     
     
         49 . A method for preparing single stranded DNA molecules, comprising:
 (a) fragmenting large or whole genomic template DNA molecules to generate a plurality of fragmented DNA molecules;   (b) ligating a first universal double stranded adaptor or a second universal adaptor to a first end of each fragmented DNA molecule and a first universal adaptor or second universal adaptor to a second end of each fragmented DNA molecule to produce a mixture of adaptor ligated DNA molecules;   (c) attaching adaptor ligated DNA molecules comprising a first double stranded universal adaptor and a second double stranded adaptor to a solid support via one strand of the first double stranded universal adaptor;   (d) washing away adaptor ligated DNA molecules which have not attached to a solid support;   (e) strand separating those adaptor ligated DNA molecules that are attached to a solid support at only one end to release a plurality of single stranded DNA molecules comprising a first single stranded universal adaptor at one end and a second single stranded adaptor at the other end; and; and   (f) isolating the single stranded DNA molecules.   
     
     
         50 . A method for delivering nucleic acid templates to a plurality of reaction centers comprising the steps of:
 (a) providing a population of nucleic acid templates;   (b) isolating each nucleic acid template from said population to a sequestering agent to form a population of sequestered nucleic acid templates;   (c) delivering said population of sequestered nucleic acid templates to said plurality of reaction centers wherein each reaction center receives one sequestered nucleic acid.   
     
     
         51 . The method of  claim 50 , wherein said isolating step comprises attaching said nucleic acid templates to a bead. 
     
     
         52 . The method of  claim 50 , wherein said isolating step comprises encapsulating said nucleic acid template in an emulsion of a water-in-oil emulsion. 
     
     
         53 . The method of  claim 52 , wherein said nucleic acid template is encapsulated with a bead and wherein the bead can bind said nucleic acid. 
     
     
         54 . The method of  claim 50 , wherein said delivering step comprises delivering said sequestered nucleic acid to a plurality of reaction centers, wherein each reaction center is a well on a picotiter plate. 
     
     
         55 . The method of  claim 1 ,  2  or  49  further comprising the step of attaching the isolated single stranded molecules each individually to a solid support.