Methods and compositions for evaluating genetic markers

1. A method for capturing and sequencing a nucleic acid target, the method comprising:

providing a sample comprising human nucleic acid;

introducing a probe set comprising a plurality of different molecular inversion probes, wherein each probe is designed to capture one of a plurality of overlapping sub-regions of a target on a strand of the nucleic acid;

hybridizing the probes to the strand;

converting the hybridized probes into covalently-closed circularized probes, thereby capturing the overlapping sub-regions of the target, wherein at least two different molecular inversion probes capture any given single base along the target;

isolating the circularized probes;

amplifying the isolated circularized probes to generate amplicons;

sequencing the amplicons to generate sequence reads of the captured sub-regions; and

analyzing the sequence reads to detect a mutation in said target nucleic acid specific for a disease.

2. The method of claim 1 , wherein the amplification step requires a single set of primers.

3. The method of claim 1 , wherein the disease is selected from the group consisting of: Familial hyperinsulinism, Canavan disease, Maple Syrup Urine disease, Bloom syndrome, Cystic fibrosis, Dihydrolipoamide dehydrogenase deficiency, Fanconi anemia, Glycogen Storage disease, Tay-Sachs diseases, Familial dysautonomia, Mucolipidosis, Usher syndrome, and Neimann-Pick disease.

4. The method of claim 1 , wherein the plurality of sub-regions comprises coding and non-coding regions of the human nucleic acid.

5. The method of claim 1 , wherein the probe set is a plurality of probe sets, each probe set specific for a different target nucleic acid and wherein each target nucleic acid is specific for a different disease.

6. The method of claim 1 , wherein said analyzing step comprises:

assembling the sequence reads into contigs; aligning the contigs to a reference; and

aligning the sequence reads to the contigs to determine mapping and variant information of the sequence reads relative to said reference to obtain a genotype at said mutation.

7. The method of claim 1 , wherein the overlapping sub-regions are at least 10 base-pairs in length.

8. The method of claim 1 , wherein the plurality of overlapping sub-regions of the target nucleic acid are from a gene selected from the list consisting of: ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (ABCC8); aspartoacylase (ASPA); branched chain keto acid dehydrogenase E1, alpha polypeptide (BCKDHA); branched chain keto acid dehydrogenase E1, beta polypeptide (BCKDHB); Bloom Syndrome, RecQ Helicase-Like (BLM); cystic fibrosis transmembrane conductance regulator (CFTR), clarin 1 (CLRN1); dihydrolipoamide dehydrogenase (DLD); Fanconi anemia, complementation group C (FANCC); glucose-6-phosphatase, catalytic subunit (G6PC); hexosaminidase A alpha polypeptide (HEXA); kinase complex-associated protein (IKBKAP); mucolipin 1 (MCOLN1); protocadherin-related 15 (PCDH15); and sphingomyelin phosphodiesterase 1, acid lysosomal (SMPD1).

9. The method of claim 1 , wherein each probe is a molecular inversion probe (MIP) that comprises an oligonucleotide with

a first targeting arm at a 5′ end and

a second targeting arm at a 3′ end, wherein the first and second targeting arms are capable of specifically hybridizing to a first and second regions flanking one of the overlapping subregions.

10. The method of claim 9 , wherein each MIP includes a differentiator tag between the first and second targeting arms, wherein the differentiator tags are different across the probe set.

11. The method of claim 10 , wherein each differentiator tag provides a unique sequence tag to an individual target molecule captured by one of the plurality of different molecular inversion probes.

12. The method of claim 11 , wherein, after the amplifying and sequencing steps, the differentiator tags distinguish independent target molecules from one another.

13. The method of claim 11 , wherein the enzymatic removal uses an exonuclease.

14. The method of claim 9 , wherein the isolating step comprises enzymatic removal of non-circularized nucleic acid.