Methods for genome assembly and haplotype phasing
View Patent ↗The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure further provides methods for haplotype phasing and meta-genomics assemblies.
1. A method for haplotype phasing, the method comprising:
providing a cell comprising chromosomes;
crosslinking DNA within the chromosomes to form crosslinked DNA;
fragmenting the crosslinked DNA to form fragmented crosslinked DNA;
ligating two regions of the fragmented crosslinked DNA that are in close physical proximity to create ligated DNA comprising a ligation product, wherein the two regions comprise an intra-chromosomal pair;
shearing the ligated DNA to generate a plurality of smaller genomic fragments;
sequencing the plurality of smaller genomic fragments to generate sequencing reads;
identifying at least one heterozygous site in the sequencing reads; and
using the sequencing reads to determine haplotype phasing of the at least one heterozygous site.
2. The method of claim 1 , further comprising isolating the chromosomes before the crosslinking.
3. The method of claim 1 , further comprising purifying DNA prior to the sequencing.
4. The method of claim 3 , wherein the purifying comprises pulling down junction-containing fragments.
5. The method of claim 1 , further comprising, after the fragmenting, marking ends of the fragmented crosslinked DNA.
6. The method of claim 5 , wherein the marking comprises filling in ends of the fragmented crosslinked DNA with nucleotides comprising one or more markers.
7. The method of claim 6 , wherein the one or more markers are usable in a pull down.
8. The method of claim 7 , wherein the one or more markers comprise biotin.
9. The method of claim 1 , wherein the fragmenting comprises digesting the crosslinked DNA with a restriction enzyme.
10. The method of claim 1 , wherein the crosslinking comprises contacting DNA with formaldehyde.
11. The method of claim 1 , wherein the ligating comprises contacting the fragmented crosslinked DNA with a ligase.
12. The method of claim 1 , wherein the sequencing comprises paired-end sequencing.
13. The method of claim 1 , wherein the cell is a human cell.
14. The method of claim 1 , wherein the cell is diploid.