Method for detecting cystic fibrosis

Abstract

The present invention relates to methods for simultaneously determining the presence or absence of mutations, deletions, duplications and single nucleotide polymorphisms in a cystic fibrosis transmembrane regulator (CFTR) nucleic acid. Oligonucleotide primers and kits used to amplify regions of a CFTR nucleic acid for high throughput, massively parallel sequencing and methods of determining an individual's cystic fibrosis status are also disclosed.

That which is claimed is: 
     
         1 . A method for determining the nucleotide sequence of a sample CFTR nucleic acid comprising:
 (a) producing an adapter-tagged amplicon library by amplifying multiple target segments of the sample CFTR nucleic acid, wherein each target segment is amplified with a pair of oligonucleotide primers, wherein at least one primer of the primer pair is selected from the group consisting of SEQ ID NOS: 3-174; and   (b) determining the nucleotide sequences of the target segments by sequencing the amplicons in the amplicon library using high throughput massively parallel sequencing.   
     
     
         2 . The method of  claim 1 , wherein the multiple target segments are amplified by PCR. 
     
     
         3 . The method of  claim 1 , wherein the sample CFTR nucleic acid is at least one nucleic acid selected from the group consisting of genomic DNA, mRNA and cDNA. 
     
     
         4 . The method of  claim 1 , wherein an adapter sequence is ligated to one of both ends of each amplicon. 
     
     
         5 . The method of  claim 1 , wherein at least one primer of the primer pair is ligated to a sequencing adapter sequence prior to amplification. 
     
     
         6 . The method of  claim 1 , wherein the amplicons are labeled with an index label that indicates the sample source from which the amplicon is generated. 
     
     
         7 . The method of  claim 6 , wherein the index label is an oligonucleotide. 
     
     
         8 . The method of  claim 1 , wherein the multiple target segments of the sample CFTR nucleic acid, together, span the CFTR coding region and all intron/junctions. 
     
     
         9 . The method of  claim 8 , wherein the multiple target segments further span about 1000 nucleotides of the promoter region immediately upstream of the first exon. 
     
     
         10 . The method of  claim 9 , wherein the multiple target segments further span about 200 to 350 nucleotides immediately downstream of the CFTR sequence. 
     
     
         11 . The method of  claim 1 , wherein the at least one primer of the primer pair is selected from the group consisting of SEQ ID NOs: 9-54, 61-106, 109-140, and 143-174. 
     
     
         12 . The method of  claim 1 , wherein the high throughput massively parallel sequencing involves a read depth approach. 
     
     
         13 . A kit comprising an oligonucleotide primer selected from the group consisting of SEQ ID NO: 3-174, wherein the primer further comprises a fluorescent label. 
     
     
         14 . The kit of  claim 13 , wherein the oligonucleotide primer is selected from the group consisting of SEQ ID NO: 9-54, 61-106, 109-140, and 143-174. 
     
     
         15 . The kit of  claim 13 , wherein the oligonucleotide primer is ligated to a sequencing adapter sequence. 
     
     
         16 . The kit of  claim 15 , wherein the sequencing adapter sequence comprises SEQ ID NO: 1. 
     
     
         17 . The kit of  claim 15 , wherein the sequencing adapter sequence comprises SEQ ID NO: 2.