Whole-genome haplotype reconstruction
AssigneeLudwig Institute for Cancer Research
InventorsBing Ren, Siddarth Selvaraj, Jesse Dixon, Anthony Schmitt
Life of this patent
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Abstract
The present invention relates to methods for haplotype determination and, in particular, haplotype determination at the whole genome level as well as targeted haplotype determination.
Claims25
-36. (canceled)
A method of detecting a genetic variant associated with disease in a eukaryotic organism, the method comprising:
- providing a cell of the organism that contains a set of chromosomes having genomic DNA comprising a genetic variant associated with disease;
- incubating the cell or the nuclei thereof with a fixation agent for a period of time to allow crosslinking of the genomic DNA in situ and thereby to form crosslinked genomic DNA;
- fragmenting the crosslinked genomic DNA;
- ligating the proximally located crosslinked and fragmented genomic DNA to form a proximally ligated complex having a first genomic DNA fragment and a second genomic DNA fragment;
- shearing the proximally ligated complex to form proximally-ligated DNA fragments;
- obtaining a plurality of the proximally-ligated DNA fragments to form a library;
- sequencing the plurality of the proximally-ligated DNA fragments to obtain a plurality of sequence reads and
- assembling the plurality of sequence reads to construct a haplotype for one or more of the chromosomes;
- and
- detecting whether a genetic variant associated with disease is present in the eukaryotic organism.
The method of claim 1, wherein the first genomic DNA fragment and the second genomic DNA fragment are on the same chromosome.
The method of claim 1, wherein the genetic variant is associated with cancer.
The method of claim 1, wherein the genetic variant is a marker for resistance to cancer treatment drugs.
The method of claim 1, wherein the steps of obtaining a plurality of the proximally-ligated DNA fragments to form a library and of sequencing the plurality of the proximally-ligated DNA fragments to obtain a plurality of sequence reads comprise:
- contacting the proximally-ligated DNA fragments with one or more oligonucleotides that hybridize to pre-selected regions of a subset of the proximally-ligated fragments to provide a subset of proximally-ligated fragments hybridized to the oligonucleotides, separating the subset of proximally-ligated fragments from the oligonucleotides;
- sequencing the subset of proximally-ligated DNA fragments to obtain a plurality of sequence reads.
The method of claim 1, further comprising isolating the cell nuclei from the cell before the incubating step.
The method of claim 1, further comprising purifying ligated genomic DNA before the fragmenting step.
The method of claim 1, further comprising after the fragmenting step:
- labeling the first genomic DNA fragment or the second genomic DNA fragment with a marker;
- joining the first genomic DNA fragment and the second genomic DNA fragment so that the maker is there between to form a labeled chimeric DNA molecule;
- and
- shearing the labeled chimeric DNA molecule to form labeled, proximally-ligated DNA fragments.
The method of claim 1, wherein the fragmenting step is carried out by digesting the ligated genomic DNA with a restriction enzyme to form digested genomic DNA fragments.
The method of claim 1, wherein the fixation agent comprises formaldehyde, glutaraldehyde, or formalin.
The method of claim 1, wherein the sequencing is carried out using pair-end sequencing of pair end sequencing fragments.
The method of claim 1, wherein, for each chromosome, the library contains at least 15×sequence coverage.
The method of claim 1, wherein the organism is a fungus, a plant, an animal, a mammal, or a mammalian embryo.
The method of claim 1, wherein the cell is a diploid cell or an aneuploid cell.
The method of claim 1, wherein the assembling is carried out in conjunction with local conditional phasing.
Dependencies are inferred from the claim text.