Classification of nucleic acid templates

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

What is claimed is: 
     
         1 . A method of determining a sequence of a double-stranded nucleic acid sample and a position of at least one modified base in the sequence, comprising:
 a) locking the forward and reverse strands together to form a circular pairlocked molecule;   b) obtaining sequence data of the circular pair-locked molecule via single molecule sequencing, wherein the sequence data comprises sequences of the forward and reverse strands of the circular pair-locked molecule;   c) determining the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands of the circular pair-locked molecule;   d) obtaining sequencing data of the circular pair-locked molecule via single molecule sequencing, wherein at least one nucleotide analog that discriminates between a base and its modified form is used to obtain sequence data comprising at least one position wherein the at least one differentially labeled nucleotide analog was incorporated; and   e) determining the positions of modified bases in the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands.   
     
     
         2 . A method of determining a sequence of a double-stranded nucleic acid sample and a position of at least one modified base in the sequence, comprising:
 a) locking the forward and reverse strands of the nucleic acid sample together to form a circular pair-locked molecule;   b) obtaining sequence data of the circular pair-locked molecule via single molecule sequencing, wherein at least one nucleotide analog that discriminates between a base and its modified form is used to obtain sequence data comprising at least one position wherein the at least one differentially labeled nucleotide analog was incorporated; and   c) determining the sequence of the double-stranded nucleic acid sample and the position of the at least one modified base in the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands of the circular pair-locked molecule.   
     
     
         3 . A method of determining a sequence of a double-stranded nucleic acid sample and a position of at least one modified base in the sequence, comprising:
 a) linking the forward and reverse strands together to form a circular template molecule comprising a double stranded segment joined at both ends by linking oligonucleotides;   b) obtaining sequence data of the circular template molecule via single molecule sequencing, wherein the sequence data comprises sequences of the forward and reverse strands of the circular template molecule, and wherein at least one nucleotide analog that discriminates between a base and its modified form is used to obtain sequence data comprising at least one position wherein at least one nucleotide analog that discriminates between a base and its modified form was incorporated;   c) determining the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands of the circular template molecule;   and   d) determining the positions of modified bases in the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands.   
     
     
         4 . A method of determining a sequence of a double-stranded nucleic acid sample and a position of at least one modified base in the sequence, comprising:
 a) linking the forward and reverse strands of the nucleic acid sample together to form a circular template molecule comprising a double stranded segment joined at both ends by linking oligonucleotides;   b) obtaining sequence data of the circular template molecule via single molecule sequencing, wherein at least one nucleotide analog that discriminates between a base and its modified form is used to obtain sequence data comprising at least one position wherein at least one nucleotide analog that discriminates between a base and its modified form was incorporated; and   c) determining the sequence of the double-stranded nucleic acid sample and the position of the at least one modified base in the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands of the circular template molecule.