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 sequencing a target nucleic acid without the use of nanodrops comprising:
(a) combining in a single mixture (i) first fragments of the target nucleic acid, and (ii) a population of beads, wherein each bead comprises oligonucleotides immobilized thereon, said oligonucleotides comprising a tag-containing sequence, wherein each tag-containing sequence comprises a tag sequence, wherein the oligonucleotides immobilized on the same individual bead comprise the same tag-containing sequence and a majority of beads have different tag sequences;
(b) introducing into a plurality of the first fragments multiple copies of a tag sequence, wherein the multiple copies are from a single bead per first fragment;
(c) producing a plurality of subfragments from the plurality of first fragments, wherein a plurality of the subfragments comprise one or more tags;
(d) sequencing the plurality of subfragments to produce a plurality of sequence reads.
2. The method of claim 1 wherein producing the plurality of subfragments comprises performing an amplification reaction to produce a plurality of amplicons from each first fragment.
3. The method of claim 1 wherein the tag-containing sequences comprise transposon ends, the method comprising combining the first fragments and the tag-containing sequences under conditions that are suitable for transposition of the tag-containing sequences into the first fragments.
4. The method of claim 1 wherein the target nucleic acid is a complex nucleic acid.
5. The method of claim 1 wherein the target nucleic acid is genomic DNA of an organism.
6. The method of claim 5 comprising determining a haplotype of the genome.
7. The method of claim 5 wherein the genomic DNA is from a diploid organism.
8. The method of 7 wherein the organism is a human.
9. The method of claim 8 wherein the genomic DNA comprises DNA from circulating fetal cells or circulating tumor cells.
10. The method of claim 1 wherein the target nucleic acid is a microbiome comprising bacterial DNA from a mixture of bacteria.
11. The method of claim 1 comprising the additional steps:
(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.
12. The method of claim 1 wherein the tag-containing sequences comprise transposon ends, the method comprising combining the plurality of first fragments with the population of beads under conditions that are suitable for transposition of the tag sequences into the first fragments.
13. The method of claim 1 wherein the first fragments comprise genomic DNA sequences and adaptor sequences.
14. The method of claim 13 wherein the tag-containing sequences comprise a tag sequence and a sequence complementary to the adaptor sequences.
15. The method of claim 14 wherein, prior to combining the plurality of first fragments with the population of beads, the first fragments are prepared by nicking on both strands.
16. The method of claim 15 further comprising ligating adaptor sequences into each strand at nicks.
17. The method of claim 14 wherein the first fragments are partially double stranded.
18. The method of claim 17 wherein the partially double stranded first fragments are prepared by denaturing fragments of the target nucleic to produce single strands, annealing random primers to the single strands, extending the primers to produce a partially double stranded first fragment, and ligating adaptors to the extended primers.
19. The method of claim 5 wherein prior to combining the population of beads and the first fragments of genomic DNA, the genomic DNA is treated by transposon insertion and fragmenting.