Multiple tagging of individual long DNA fragments
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 generating tagged sequence reads comprising:
(a) combining:
(i) a plurality of long fragments of a target nucleic acid, wherein the fragments are from about 5 kilobases to about 750 kilobases in length;
(ii) a population of beads, wherein each bead comprises multiple copies of a single polynucleotide comprising a tag-containing sequence, wherein said tag-containing sequence comprises a tag sequence and a transposon end sequence, and wherein different beads comprise different tag sequences; and
(iii) a transposase,
in a single reaction vessel under conditions in which, for a plurality of beads, the tag sequence associated with an individual bead is transposed into multiple sites of an individual long fragment;
(b) producing a plurality of subfragments of the long fragments; and,
(c) sequencing subfragments from (b) to produce a plurality of sequence reads including tag sequences.
2. The method of claim 1 wherein said tag-containing sequences comprise a tag sequence and producing tagged subfragments comprises performing an amplification reaction to produce a plurality of amplicons.
3. The method of claim 1 wherein the target nucleic acid is a complex nucleic acid.
4. The method of claim 3 wherein the target nucleic acid is a genome of an organism.
5. The method of claim 1 wherein the length of the tag sequence is from 8 to 12 nucleotides.
6. The method of claim 5 comprising determining a haplotype of the genome.
7. The method of claim 1 wherein the tag-containing sequences comprise a region of degenerate sequence that serves as a tag sequence, a known sequence that is a PCR primer binding site, and a known sequence that is a transposon end sequence.
8. The method of claim 1 further comprising mapping and assembling the sequence reads into genome sequence.
9. The method of claim 1 further comprising assembling the sequence reads to produce an assembled sequence of a long fragment.
10. The method of claim 1 wherein each bead comprises hundreds of copies of the polynucleotide comprising a unique tag-containing sequence.
11. The method of claim 10 wherein each bead comprises thousands of copies of the polynucleotide comprising a unique tag-containing sequence.
12. The method of claim 1 wherein tag sequences of at least some beads are transpose into more than one long fragment.
13. The method of claim 1 wherein for a majority of long fragments tagged at multiple sites, said sites are separated from each other by an average spacing in the range of 100 bp to 5,000 bp.