SINGLE TUBE BEAD-BASED DNA CO-BARCODING FOR ACCURATE AND COST-EFFECTIVE SEQUENCING, HAPLOTYPING, AND ASSEMBLY

1 . A method of preparing a sequencing library for sequencing a target nucleic acid without the use of nanodrops comprising:

(a) transposing an insertion sequence into first fragments of the target nucleic acid, wherein the insertion sequence comprises a hybridization sequence, and wherein the transposing produces nicks in the first fragments, wherein optionally the target nucleic acid is not amplified prior to step (a);

(b) combining in a single mixture (i) the first fragments of the target nucleic acid from (a), (ii) a splint oligonucleotide, and (iii) a population of beads, wherein each bead comprises capture oligonucleotides immobilized thereon, wherein optionally the capture oligos are connected to beads at the 5′ terminus end,

wherein optionally the population of beads comprises, in aggregate, at least 100,000 different tag sequences or at least 1 million different tag sequences,

wherein, optionally, on average, the spacing of barcoded capture oligos on the beads is <10 nm, <15 nm, <20 nm, <30 nm, <40 nm, or 50 nm

wherein said capture oligonucleotides comprising

1) a tag (or barcode)-containing sequence, wherein each tag (or barcode)-containing sequence comprises a barcode sequence, which is optionally a tripartate barcode sequence, wherein the oligonucleotides immobilized on the same individual bead comprise the same tag (or barcode)-containing sequence and a majority of beads have different tag (or barcode)-sequences,

2) a common sequence complementary to at least a portion of a splint oligonucleotide, where a second portion of the splint oligonucleotide is complementary to at least a portion of the hybridization sequence;

3) optionally a first PCR primer annealing site;

(c) ligating capture oligonucleotides of individual beads to inserted hybridization sequences of individual first fragments.

2 . The method of claim 1 wherein the capture oligonucleotides comprise the first PCR primer annealing site further comprising ligating a 3′ branch ligation adaptor oligonucleotide to the first fragments at the nicks, wherein the ligating an adaptor oligonucleotide is a 3′ branch ligation, and wherein the adaptor oligonucleotide comprises a second PCR primer annealing site.

3 . The method of claim 2 wherein the 3′ branch ligation adaptor oligonucleotide is a blunt end adaptor and the 3′ branch ligation comprises the covalent joining of the 5′ phosphate from the blunt-end adapter to the recessed 3′ hydroxyl a the nicks of the first fragments.

4 . The method of claim 1 wherein the first PCR primer annealing site and the second PCR primer annealing site have different sequences.

5 . The method of claim 1 , wherein the 3′ branch ligation adaptor oligonucleotide comprises a barcode sequence, with is optionally a sample barcode sequence.

6 . The method of claim 1 , wherein in the step of transposing the insertion sequence into the first fragments the transposase enzyme remains bound to the first fragments.

7 . The method of claim 1 , the method comprising removing the transposase from the first fragments thereby producing subfragments,

wherein optionally the size of the subfragments are in the range of 200 to 2,000 bp in length

wherein optionally the method comprises sequencing at least some of the subfragments or the method to produce sequence reads,

wherein optionally the sequence reads with the same barcode sequence are from the same first fragment,

wherein optionally the size of the subfragments are in the range of 200 to 2,000 bp in length,

wherein the size of the subfragments are in the range of 200 to 2,000 bp in length>10% or >20%, >40%, >50%, >60% of subfragments of long fragments of the subfragments are sequenced, and

wherein optionally >50%, >70%, >80%, >90%, >95% of DNA first fragments are barcoded with the unique barcode.

8 . The method of claim 7 comprising amplifying the subfragments to produce amplicons, wherein optionally the method comprises sequencing the amplicons to produce sequence reads wherein sequence reads with the same barcode sequence are from the same first fragment.

9 - 10 . (canceled)

11 . The method of claim 1 wherein two different transposons are inserted into the first fragments.

12 . The method of claim 1 , wherein the target nucleic acid is genomic DNA, optionally human genomic DNA, optionally from a eukaryote, optionally a mammal,

wherein optionally the genomic DNA is from a single individual organism, such as a single human subject, and

wherein optionally the target nucleic acid is from a prokaryote or a heterogeneous population of prokaryotes such as a microbiome from an individual.

13 - 15 . (canceled)

16 . The method of claim 1 , wherein the beads comprise at least 100,000 copies of the capture oligonucleotide, or comprises at least 400,000 copies of the capture oligonucleotide, and wherein optionally more than 1 billion beads are used in the reaction.

17 . (canceled)

18 . The method of claim 8 wherein, prior to the amplifying (i) at least some the capture oligonucleotides are enzymatically removed and/or at least some splint oligonucleotides are enzymatically removed and/or at least some mosaic end (ME) sequences are enzymatically removed,

wherein optionally at least some capture oligonucleotides are enzymatically removed using exonuclease, optionally exonuclease I or III or both.

19 . (canceled)

20 . The method of claim 1 , wherein the capture oligonucleotides and/or the splint oligonucleotides comprise uracil, optionally, wherein treatment with a Uracil-DNA Glycosylase (UDG) degrades uracil-containing oligonucleotides.

21 - 23 . (canceled)

24 . The method of claim 1 , wherein >50%, >70%, >80% >90% of subfragments in a fragment are ligated to barcode oligo.

25 - 28 . (canceled)

29 . The method of claim 8 , comprising the additional steps:

(e) assigning a majority of the sequence reads to corresponding first fragments; and

(f) assembling the sequence reads to produce an assembled sequence of the target.

30 . The method of claim 1 , wherein the majority of first fragments in Step(a) are longer than 20 kb, or longer than 50 kb, or longer than 100 kb, or in the range of from 50 kilobases to 200 kilobases in length.

31 - 33 . (canceled)

34 . The method of claim 1 , wherein the plurality of first fragments are from a single cell, or from 1-100 eukaryotic cells, or from 2-10 eukaryotic cells, or from 3-30 human cells.

35 - 37 . (canceled)

38 . The method claim 1 , wherein the single vessel or mixture contains 5-1000 (e.g., 5-20) genome equivalents of human DNA.

39 - 43 . (canceled)

44 . A composition comprising (i) a plurality of first fragments of a target nucleic acid, (ii) a population of beads comprising capture oligonucleotides hybridized thereon wherein each bead comprises a plurality of oligonucleotides with the same barcode, and different beads in the population comprise different barcodes, wherein the first fragments comprise transposon integrated DNA, said transposons comprising a common hybridization sequence; and (iii) a splint oligonucleotide complementary to the hybridization sequence.

45 . A composition comprising a population of beads comprising clonal capture oligonucleotides attached thereon wherein each bead comprises a plurality of capture oligonucleotides with the same barcode, and different beads in the population comprise different barcodes,

wherein a plurality of individual beads comprises a transposon inserted target DNA linked to the bead; wherein the link is the ligation of a plurality of capture oligonucleotides on the individual bead to a plurality of hybridization sequences in the a transposon inserted target DNA; and the composition comprises at least one of

i) an exonuclease;

ii) a DNA polymerase;

iii) a Uracil-DNA Glycosylase;

iv) a ligase;

iv) a 3′ Branch Ligation Adaptor.