Multiple tagging of individual long dna fragments

Abstract

This disclosure provides methods and compositions for tagging long fragments of a target nucleic acid for sequencing and analyzing the resulting sequence information in order to reduce errors and perform haplotype phasing, for example.

1 . A method of sequencing a target nucleic acid comprising:
 (a) combining in a single reaction vessel (i) a plurality of long fragments of the target nucleic acid, and (ii) a population of polynucleotides, wherein each polynucleotide comprises a tag and a majority of the polynucleotides comprise a different tag;   (b) introducing into a majority of the long fragments tag-containing sequences from said population of polynucleotides to produced tagged long fragments, wherein each of the tagged long fragments comprises a plurality of the tag-containing sequences at a selected average spacing, and each tag-containing sequence comprises a tag;   (c) producing a plurality of subfragments from each tagged long fragment, wherein each subfragment comprises one or more tags;   (d) sequencing the subfragments to produce a plurality of sequence reads;   (e) assign a majority of the sequence read to corresponding long fragments; and   (f) assembling the sequence reads to produce an assembled sequence of the target   
     
     
         2 . The method of  claim 1  wherein producing the subfragments comprises performing an amplification reaction to produce a plurality of amplicons from each long fragment. 
     
     
         3 . The method of  claim 2  wherein each amplicon comprises a tag from each of the adjacent introduced sequences and a region of the long fragment between the adjacent introduced sequences. 
     
     
         4 . The method of  claim 1  comprising combining the long fragments with an excess of the population of tag-containing sequences. 
     
     
         5 . The method of  claim 1  comprising combining the long fragments with the tag-containing solution under conditions that are suitable for introduction of a single tag-containing sequence into a majority of the long fragments. 
     
     
         6 . The method of  claim 1  comprising combining the long fragments with the tag-containing solution under conditions that are suitable for introduction of a different tag-containing sequences into a majority of the long fragments. 
     
     
         7 . The method of  claim 1  wherein the population of tag-containing sequences comprises a population of beads, wherein each bead comprises multiple copies of a single tag-containing sequence. 
     
     
         8 . The method of  claim 1  wherein the population of tag-containing sequences comprises a population of concatemers, each concatemer comprising multiple copies of a single tag-containing sequence. 
     
     
         9 . The method of  claim 1  wherein the tag-containing sequences comprise transposon ends, the method comprising combining the long fragments and the tag-containing sequences under conditions that are suitable for transposition of the tag-containing sequences into each of the long fragments. 
     
     
         10 . The method of  claim 1  wherein the tag-containing sequences comprise a hairpin sequence. 
     
     
         11 . The method of  claim 1  wherein the target nucleic acid is a complex nucleic acid. 
     
     
         12 . The method of  claim 1  wherein the target nucleic acid is a genome of an organism. 
     
     
         13 . The method of  claim 12  comprising determining a haplotype of the genome.