Back to results
US 12,006,538 B2Granted
3-D genomic region of interest sequencing strategies
AssigneeCergentis B.V.
InventorsWouter Leonard de Laat, Max Jan van Min
Life of this patent
Estimating the term…
Abstract
The invention relates to methods for determining the sequence of a genomic region of interest comprising a target nucleotide sequence comprising, fragmenting a crosslinked DNA, ligating the fragmented cross linked DNA, reversing the crosslinking and determining at least part of the sequences of ligated DNA fragments which comprise a target nucleotide sequence.
Claims16
A method of generating one or more contigs of one or more genomic regions of interest, the method comprising:
- a) providing a DNA sample comprising protein and at least 1000 DNA molecules each comprising the one or more genomic regions of interest,
- wherein each genomic region of interest comprises
- one or more target polynucleotide sequences and
- DNA sequence in addition to the one or more target polynucleotide sequences, and
- wherein each of the one or more target polynucleotide sequences is of sufficient length to distinguish a nucleotide sequence comprising the target polynucleotide sequence from the other nucleotide sequences in the DNA molecules,
- b) treating the DNA sample with formaldehyde to crosslink the one or more genomic regions of interest,
- c) fragmenting the crosslinked genomic regions to generate crosslinked DNA fragments of the one or more genomic regions of interest having ligatable ends, wherein a set of the crosslinked DNA fragments comprises at least one target nucleotide sequence,
- d) ligating the crosslinked DNA fragments to produce ligated DNA products of at least 100 bp in length and comprising at least three overlapping and/or directly flanking DNA fragments from the one or more genomic regions of interest in addition to a DNA fragment comprising the one or more target polynucleotide sequences,
- e) reversing the crosslinking;
- f) optionally, specifically amplifying the ligated DNA products to produce amplification products,
- g) sequencing the ligated products, or amplification products thereof, so as to generate at least 17578 sequences of at least 20 base pairs in length, wherein the sequences comprise overlapping and/or directly flanking sequences of the at least three overlapping and/or directly flanking DNA fragments, wherein at least 4.9% of the nucleotides directly corresponding to the individual nucleotides of each of the one or more genomic regions of interest has been sequenced at least 20 times, and wherein the sequences are sufficient to build a contig of at least one of the one or more genomic regions of interest, and
- h) aligning, with a computer program, the at least 17578 sequences to one or more reference sequences comprising at least one of the one or more genomic regions of interest to generate one or more contigs of at least one of the one or more genomic regions of interest, wherein the one or more contigs are generated utilizing the overlapping and/or directly flanking sequences of the at least three overlapping and/or directly flanking DNA fragments.
9 more elements
A method of generating one or more contigs of one or more genomic regions of interest, the method comprising:
- a) providing a DNA sample comprising protein and at least 1000 DNA molecules each comprising the one or more genomic regions of interest,
- wherein each genomic region of interest comprises
- one or more target polynucleotide sequences and
- DNA sequence in addition to the one or more target polynucleotide sequences, and
- wherein each of the one or more target polynucleotide sequences is of sufficient length to distinguish a nucleotide sequence comprising the target polynucleotide sequence from the other nucleotide sequences in the DNA molecules,
- b) treating the DNA sample with formaldehyde to crosslink the one or more genomic regions of interest,
- c) fragmenting the crosslinked genomic regions to generate crosslinked DNA fragments of the one or more genomic regions of interest having ligatable ends, wherein a set of the crosslinked DNA fragments comprises at least one target nucleotide sequence,
- d) ligating the crosslinked DNA fragments to produce ligated DNA products of at least 100 bp in length and comprising at least three overlapping and/or directly flanking DNA fragments from the one or more genomic regions of interest in addition to a DNA fragment comprising the one or more target polynucleotide sequences,
- e) reversing the crosslinking;
- f) optionally, specifically amplifying the ligated DNA products to produce amplification products,
- g) sequencing the ligated products, or amplification products thereof, so as to generate at least 17578 sequences of at least 20 base pairs in length, wherein the sequences comprise overlapping and/or directly flanking sequences of the at least three overlapping and/or directly flanking DNA fragments, wherein at least 4.9% of the nucleotides directly corresponding to the individual nucleotides of each of the one or more genomic regions of interest has been sequenced at least 20 times, and wherein the sequences are sufficient to build a contig of at least one of the one or more genomic regions of interest, and
- h) aligning, with a computer program, the at least 17578 sequences to generate one or more contigs of at least one of the one or more genomic regions of interest, wherein the one or more contigs are assembled utilizing the overlapping and/or directly flanking sequences of the at least three overlapping DNA fragments and wherein the overlaps of the at least three overlapping DNA fragments are sufficient to assemble the one or more contigs.
9 more elements
Dependencies are inferred from the claim text.