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US 12,742,195 B2Granted
Genotyping by sequencing
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsGoncalo Abecasis, Mathew Barber, William Salerno
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Abstract
The present disclosure provides methods for manufacturing nucleic acid probes for genotyping by sequencing, methods for genotyping a DNA sample by sequencing using a set of nucleic acid probes, and systems for carrying out such methods.
Claims22
A method for manufacturing nucleic acid probes for genotyping by sequencing, the method comprising:
- a) selecting a plurality of directly observed genetic variants to capture by the nucleic acid probes;
- b) eliminating low confidence variants from the plurality of directly observed genetic variants, thereby producing a filtered plurality of directly observed genetic variants;
- c) phasing the filtered plurality of directly observed genetic variants;
- d) identifying the presence or absence of one or more proxy variants for each variant within the filtered plurality of directly observed genetic variants;
- e) selecting a plurality of candidate regions of genomic DNA comprising the filtered plurality of directly observed genetic variants, wherein each candidate region of genomic DNA comprises from about 25 to about 150 bases, and comprises at least one variant among the filtered plurality of directly observed genetic variants;
- f) calculating a Quality score for each candidate region of genomic DNA that estimates the capture efficiency and alignment success of a probe;
- g) calculating a Probe score for each candidate region of genomic DNA by multiplying the Quality score by the number of variants captured by the candidate region of genomic DNA, wherein the number of variants captured by the candidate region of genomic DNA is the sum of the number of directly observed variants captured by the candidate region of genomic DNA and the number of corresponding proxy variants in different candidate regions of genomic DNA;
- h) selecting one or more candidate regions of genomic DNA having the highest Probe score for inclusion in a final set of regions of genomic DNA;
- i) repeating steps g) and h) on unselected candidate regions of genomic DNA for inclusion in the final set of regions of genomic DNA, wherein the number of variants in the unselected candidate region of genomic DNA is the sum of:
- 1) the number of directly observed variants in the unselected candidate region of genomic DNA excluding any directly observed variant within a previously selected region of genomic DNA, and 2) the number of corresponding proxy variants in different candidate regions of genomic DNA excluding any proxy variant corresponding to a directly observed variant within a previously selected region of genomic DNA, wherein steps g) and h) are repeated until a maximum number of regions of genomic DNA has been selected;
- and
- j) generating a set of nucleic acid probes complementary to the nucleic acid sequence of each of the genomic regions among the final set of regions of genomic DNA.
9 more elements
A method for genotyping a DNA sample by sequencing using a set of nucleic acid probes, the method comprising:
- a) selecting a plurality of regions of genomic DNA from the DNA sample comprising a plurality of directly observed genetic variants;
- b) identifying the set of nucleic acid probes for hybridization to the selected plurality of regions of genomic DNA;
- c) hybridizing the set of nucleic acid probes to the DNA sample to generate probe-hybridized genomic DNA;
- d) sequencing the probe-hybridized genomic DNA to produce a plurality of sequencing reads corresponding to genomic regions captured by the set of nucleic acid probes;
- e) mapping the plurality of sequencing reads to a reference genome;
- f) calling the directly observed variants present in the mapped sequencing reads obtained in step e), wherein the directly observed variants comprise variants captured by hybridization of the set of nucleic acid probes and subsequently sequenced;
- and
- g) imputing, based on the presence of directly observed variants identified in step f), unobserved variants from genomic regions of the DNA sample not captured by hybridization of the set of nucleic acid probes and not sequenced, thereby establishing a genotype of the sample DNA.
5 more elements
A system comprising:
- a data processor having a memory coupled thereto, wherein the memory comprises programs including instructions for:
- selecting a plurality of regions of genomic DNA from a DNA sample comprising a plurality of directly observed genetic variants;
- identifying a set of nucleic acid probes for hybridization to the selected plurality of regions of genomic DNA, and sending instructions to an oligonucleotide synthesizer for synthesis of the set of the nucleic acid probes;
- receiving a plurality of sequencing reads from the DNA sequencing apparatus produced from sequencing the generation of probe-hybridized genomic DNA upon hybridization of the set of the nucleic acid probes to a DNA sample, wherein the sequencing reads correspond to genomic regions captured by hybridization of the set of nucleic acid probes;
- mapping the plurality of sequencing reads to a reference genome;
- calling the directly observed variants present in the mapped sequencing reads obtained in the mapping step, wherein the directly observed variants comprise variants captured by hybridization of the set of nucleic acid probes and subsequently sequenced;
- and
- imputing, based on the presence of directly observed variants called from the mapped sequencing reads, unobserved variants from genomic regions of the DNA sample not captured by hybridization of the set of nucleic acid probes and not sequenced, thereby establishing a genotype of the sample DNA.
5 more elements
Dependencies are inferred from the claim text.