Copy Number Analysis of Genetic Locus

Abstract

Systems and methods for analyzing copy number of a target locus, detecting a disease associated with abnormal copy number of a target gene or a carrier thereof.

What is claimed is: 
     
         1 . A method of analyzing copy number of a target locus, the method comprising:
 (a) providing a plurality of biological specimens, each individual biological specimen comprising a target locus and one or more reference loci with known copy numbers;   (b) performing a plurality of biological assays, wherein each individual biological assay analyzes the target locus and the one or more reference loci in the each individual biological specimen and generates detectable signals such that the level of detectable signals for the target locus and the one or more reference loci correlates with their respective copy numbers;   (c) determining, based on the plurality of biological assays, a plurality of copy number estimates for the target locus normalized to the one or more reference loci; and   (d) assessing quality of the copy number estimates and/or statistical confidence of the copy number call, thereby determining if a copy number call can be made for the target locus.   
     
     
         2 . The method of  claim 1 , wherein the target locus comprises a gene or a portion thereof. 
     
     
         3 . The method of  claim 2 , wherein the gene or a portion thereof comprises an exon of survival motor neuron 1 (SMN1). 
     
     
         4 . The method of  claim 1 , wherein the one or more reference loci are selected from the group consisting of SMARCC1 and SUPT5H. 
     
     
         5 . The method of  claim 3 , wherein the exon of SMN1 is exon 7. 
     
     
         6 . The method of  claim 1 , wherein the biological assays at step (b) are real-time PCR assays that amplify the target locus and the one or more reference loci. 
     
     
         7 . The method of  claim 6 , wherein the detectable signals are fluorescent signals, and wherein the level of the fluorescent signals for the target locus or the one or more reference loci is detected at each amplification cycle. 
     
     
         8 . The method of  claim 7 , wherein step (c) comprises steps of
 (i) determining the difference in cycle numbers (ΔCti) between the target locus and the one or more reference loci to reach a pre-determined level of the fluorescent signals in each individual biological specimen;   (ii) generating a calibrator (ΔCt) reflecting the difference between a normal target locus and the one or more reference loci; and   (iii) determining a copy number estimate for the target locus in each individual biological specimen by normalizing the difference in the cycle numbers ΔCti determined at step (i) to the calibrator (ΔCt).   
     
     
         9 . The method of  claim 8 , wherein step (i) comprises first measuring cycle numbers (Cti) for each of the target locus and the one or more reference loci to reach the predetermined level of the fluorescent signals. 
     
     
         10 . The method of  claim 8 , wherein the calibrator (ΔCt) is defined by trimmed mean of the ΔCti between the target locus and the one or more reference loci for the plurality of biological specimens. 
     
     
         11 . The method of  claim 8 , wherein the copy number estimate for the target locus in each individual biological specimen is determined on a linear scale. 
     
     
         12 . The method of  claim 8 , wherein the copy number estimate for the target locus in each individual biological specimen is determined on a logarithmic scale. 
     
     
         13 . The method of  claim 8 , wherein the quality of the copy number estimates for the target locus is assessed based on the quality of data generated for the one or more references loci. 
     
     
         14 . The method of  claim 8 , wherein the statistical confidence is assessed by determining a measurement confidence and/or a call confidence. 
     
     
         15 . The method of  claim 1 , wherein the biological assays performed in step (b) are replicated. 
     
     
         16 . The method of  claim 15 , wherein the statistical confidence of the copy number call is determined by the calculation of a measurement confidence for replicate biological assays and a call confidence based on the plurality of copy number estimates. 
     
     
         17 . The method of  claim 15 , wherein step (d) comprises determining that the copy number call for the target locus can not be made if the call confidence is less than a predetermined threshold. 
     
     
         18 .- 27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein assessing the quality of the copy number estimates comprises generating quality control metrics based on cycle number measurements and the amplification curve slope thereof generated for the one or more reference genes. 
     
     
         29 . The method of  claim 1 , wherein assessing the quality of the copy number estimates comprises determining coefficient of variation between multiple replicate biological assays. 
     
     
         30 . The method of  claim 1 , wherein assessing the statistical confidence of the copy number call comprises determining a measurement confidence and/or a call confidence. 
     
     
         31 .- 56 . (canceled)