Methods for obtaining a sequence

Abstract

The invention generally relates to methods for obtaining a sequence, such as a consensus sequence or a haplotype sequence. In certain embodiments, methods of the invention involve determining an amount of amplifiable nucleic acid present in a sample, partitioning the nucleic acid based upon results of the determining step such that each partitioned portion includes, on average, a subset of unique sequences, sequencing the nucleic acid to obtain sequence reads, and assembling a consensus sequence from the reads.

1 .- 20 . (canceled) 
     
     
         21 . A kit for sequencing DNA fragments having blunt ends, comprising
 PCR amplification adapters configured to blunt end ligate to the DNA fragments;   quantitative PCR primers with homology to the PCR amplification adapters for quantitatively amplifying the DNA fragments;   a transposase enzyme and a plurality of transposon end complexes configured to cleave the DNA fragments into size-selected DNA fragments; and   a plurality of barcode sequences configured to be incorporated into the size-selected DNA fragments upon amplification, wherein each barcode sequence has at least two mismatches from every other barcode sequence in the plurality of barcode sequences.   
     
     
         22 . The kit of  claim 21 , wherein the PCR amplification adapters have a nucleotide sequence of SEQ ID NOs: 1, 2, 3, or 4. 
     
     
         23 . The kit of  claim 21 , wherein the quantitative PCR primers have a nucleotide sequence of SEQ ID NOs: 5 or 6. 
     
     
         24 . The kit of  claim 21 , wherein the transposase enzyme is a TN-5 transposase enzyme. 
     
     
         25 . The kit of  claim 21 , wherein the barcode sequences are part of the transposon end complexes configured to cleave the DNA fragments. 
     
     
         26 . The kit of  claim 21 , wherein the barcode sequences have no homopolymer regions. 
     
     
         27 . The kit of  claim 21 , wherein the bar code sequences are from about 5 nucleotides to about 15 nucleotides in length. 
     
     
         28 . The kit of  claim 21 , where in the bar code sequences are from about 4 nucleotides to about 7 nucleotides in length. 
     
     
         29 . The kit of  claim 21 , further comprising a ligase for ligating the barcode sequences to the size-selected DNA fragments. 
     
     
         30 . The kit of  claim 29 , wherein the ligase is a blunt-end ligase. 
     
     
         31 . The kit of  claim 21 , further comprising gel material for producing a gel to size-select the cleaved DNA fragments. 
     
     
         32 . The kit of  claim 21 , further comprising DNA capture beads for purifying the DNA fragments. 
     
     
         33 . The kit of  claim 32 , wherein the DNA capture beads comprise streptavidin-coated beads. 
     
     
         34 . The kit of  claim 21 , further comprising an amplification enzyme. 
     
     
         35 . The kit of  claim 34 , wherein the amplification enzyme is Taq DNA polymerase. 
     
     
         36 . The kit of  claim 21 , wherein the barcode sequences are part of an adapter that further comprises sequencing primer annealing sequences. 
     
     
         37 . The kit of  claim 21 , wherein the PCR amplification adaptors comprise a barcode sequence to identify amplified DNA fragments. 
     
     
         38 . The kit of  claim 37 , wherein the PCR amplification adaptors comprise the same barcode sequence such that the same barcode is attached to both sides of an amplified DNA fragment. 
     
     
         39 . The kit of  claim 21 , wherein the quantitative PCR primers comprise at least one fluorescent dye. 
     
     
         40 . The kit of  claim 39 , wherein the quantitative PCR primers comprise at least one fluorescent dye and one quenching dye.