Analysis methods
View Patent ↗The invention generally relates to methods for analyzing nucleic acids to identify novel mutations associated with diseases. In certain embodiments, methods of the invention involve obtaining nucleic acid from a subject having a disease, identifying at least one mutation in the nucleic acid, and comparing the mutation to a database of mutations known to be associated with the disease, wherein mutations that do not match to the database are identified as novel mutations.
1. A system for identifying a novel mutation associated with a disease, the system comprising:
a nucleic acid sequencer operable to
capture a target sequence, wherein capturing the target sequence comprises: annealing a probe oligo to a nucleic acid obtained from a subject having a disease to form an open-loop complex in which the target sequence is flanked by ends of the probe oligo, and filling and sealing a gap between the ends of the probe oligo with polymerase and ligase enzymes to form a covalently-closed circular molecule, and
sequence the covalently-closed circular molecule with the nucleic acid sequencer to thereby obtain a sequence of the nucleic acid; and
a computer, coupled to the nucleic acid sequencer, the computer comprising a processor coupled to a memory having instructions stored therein that when executed by the processor cause the system to:
compare the sequence of the nucleic acid from the subject to a reference sequence, thereby determining the presence of at least one spatially ambiguous mutation in the nucleic acid;
compare the spatially ambiguous mutation to a database of mutations known to be associated with the disease, wherein mutations that do not match to the database are identified as novel mutations;
calculate an equivalent insertion/deletion region for the spatially ambiguous mutation;
annotate the spatially ambiguous mutation with functional information;
retrieve an extreme lower position and an extreme upper position of the equivalent insertion/deletion region for the spatially ambiguous mutation; and
choose one of the extreme lower position and the extreme upper position of the equivalent insertion/deletion region for the spatially ambiguous mutation that is outside of a functional region, wherein said spatially ambiguous mutation is not causative of the disease.
2. The system of claim 1 , wherein the reference sequence is a consensus human sequence or a sequence from a non-diseased sample.
3. The system of claim 1 , wherein prior to forming the covalently-closed circular DNA molecule, the processor further causes the system to attach a barcode sequence to the nucleic acid.
4. The system of claim 1 , wherein the disease is cystic fibrosis.
5. The system of claim 4 , wherein the subject is Hispanic.
6. The system of claim 4 , wherein the target sequence in the nucleic acid contains a 5′ boundary of CFTR exon 10.
7. The system of claim 1 , wherein the sequence is obtained by sequencing-by-synthesis.
8. The system of claim 7 , wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.