Identifying modified bases using hemi-natural nucleic acids

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. Methods are provided for sequencing hemi-natural nucleic acids such as hemi-genomic DNA, having two complementary strands, one a natural sequence and the other a synthetic sequence. The identification of modified bases can be enhanced by comparing the sequencing information from the natural sequence, which has, for example, natural base modifications, with the synthetic sequence, which typically has no base modifications. The presence and identity of a modified base can be determined by monitoring kinetics, for example the kinetics of polymer meditated nucleic acid synthesis.

1 .- 20 . (canceled) 
     
     
         21 . A method for nanopore sequencing with reduced error comprising;
 providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a plurality of nanopores connecting the upper solution and lower solution, the nanopores sized to pass single stranded nucleic acids;   providing a voltage across the nanopores to produce a measurable current flow through the nanopores;   measuring the current through the nanopores over time as the nucleic acid templates are translated through the nanopore;   measuring the sequence of a portion of a plurality template nucleic acids as they translate through the pore using the measured current over time; wherein some of the plurality of template nucleic acids comprise the same sequence, and   wherein the sequence of some of the plurality of nucleic acids is measured under one set of reaction conditions, and the sequence of some of the plurality of nucleic acids is measured under a second set of reaction conditions, where the first and second reaction conditions each provide different error profiles, and   determining a sequence by combining the measured sequences under the first and second reaction conditions to obtain a reduced error rate than for a sequence determined under one reaction condition.   
     
     
         22 . The method of  claim 21  further comprising controlling the rate of translation of a single stranded portion of the template nucleic acid through the pore with a processive enzyme associated with a template nucleic acid. 
     
     
         23 . The method of  claim 22  wherein the processive enzyme comprises a polymerase, an exonuclease, or a helicase. 
     
     
         24 . The method of  claim 21  wherein the two reaction conditions comprise two different types of nanopores. 
     
     
         25 . The method of  claim 21  wherein the two reaction conditions comprise two different types of processive enzymes. 
     
     
         26 . The method of  claim 21  wherein the combined sequences represent sequences on a single molecule. 
     
     
         27 . The method of  claim 21  wherein the combined sequences represent sequences on different molecules. 
     
     
         28 . The method of  claim 21  wherein the change in reaction condition comprises a change in the temperature, pH, or in the level of divalent cation or a combination of these. 
     
     
         29 . The method of  claim 21  wherein the plurality of nanopores comprises an array of nanopores and the first and second reaction conditions are on different regions of the array of nanopores. 
     
     
         30 . The method of  claim 21  wherein at least one of the plurality of nanopores, the sequencing is carried out iteratively to generate redundant reaction data in a single nanopore. 
     
     
         31 . The method of  claim 21  wherein at least one of the plurality of template nucleic acids comprises template comprising a double stranded portion wherein the two strands of the double stranded portion are joined by a hairpin. 
     
     
         32 . The method of  claim 31  wherein one of the strands in the double stranded portion comprises a natural strand and the other strand comprises a synthetic strand. 
     
     
         33 . The method of  claim 32  wherein the natural strand comprises genomic DNA. 
     
     
         34 . The method of  claim 21  wherein the nanopores comprise solid state nanopores. 
     
     
         35 . The method of  claim 21  wherein the nanopores comprise biological nanopores. 
     
     
         36 . A method for nanopore sequencing with reduced error comprising;
 providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a plurality of nanopores connecting the upper solution and lower solution, the nanopores sized to pass single stranded nucleic acids, wherein the plurality of nanopores comprises at least two types of nanopores, each type of nanopore exhibiting a different error profile, and;   providing a voltage across the nanopores to produce a measurable current flow through the nanopores;   measuring the current through the nanopores over time as the nucleic acid templates are translated through the nanopore;   measuring the sequence of a portion of a plurality template nucleic acids as they translate through the pore using the measured current over time;   wherein for some of the nanopores, template nucleic acids having the same sequence are sequenced in both of the types of nanopores, and   determining a sequence by combining the sequences measured in one type of nanopore and the sequence measured in the other type of nanopore to obtain a reduced error rate compared to measuring the sequence in a single nanopore.   
     
     
         37 . The method of  claim 36  further comprising controlling the rate of translation of a single stranded portion of the template nucleic acid through the pore with a processive enzyme associated with a template nucleic acid. 
     
     
         38 . The method of  claim 36  wherein at least one of the plurality of nanopores, the sequencing is carried out iteratively to generate redundant reaction data in a single nanopore. 
     
     
         39 . The method of  claim 36  wherein at least one of the plurality of template nucleic acids comprises template comprising a double stranded portion wherein the two strands of the double stranded portion are joined by a hairpin. 
     
     
         40 . The method of  claim 39  wherein one of the strands in the double stranded portion comprises a natural strand and the other strand comprises a synthetic strand. 
     
     
         41 . The method of  claim 40  wherein the natural strand comprises genomic DNA.