Improving long term outcomes in kidney transplantation remains a challenge. Transplant failure within the first year has been reduced by the development of new immunosuppressive drugs and advanced surgical techniques and a lasting survival benefit was expected 
. The reality has been disappointing. While the rate of transplant loss within five years has significantly improved in the modern era, the long term attrition rate has remained largely unchanged 
This study follows the report of a risk variant in the CAV1
gene that associates with poor transplant survival 
and is the first to investigate the effect of variation in the CAV2
gene on kidney transplant outcomes. SNPs at the CAV2
locus were investigated and a trend towards association was suggested between rs13221869 in the recipient genome and transplant survival. There was no association with recipient survival or acute rejection. An attempt to replicate this association in an ethnically similar kidney transplant recipient-donor population using an alternative technology failed to genotype two alleles for this SNP.
Direct capillary sequencing was subsequently employed to investigate the CAV2
gene in detail. Initial attempts to sequence the fragment encompassing rs13221869 failed (base position 5039, Pf8, Pr8, ) and an unusual degree of optimisation was required. This study highlights not only the need for validation of interesting SNP associations using an alternative technology, but also that future genetic studies of CAV2
in particular warrant careful consideration. For example, our original Sequenom data and 3730 sequencing showed a C/T variant at base position 5039, which is the reported position of rs13221869. However, the use of a longer sequencing fragment that reads through the difficult region revealed this ‘SNP’ was due to misalignment of bases. This SNP (rs13221869) was originally identified via large-scale sequence comparisons and has not been confirmed by genotyping or population frequency data (www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=13221869
Repetitive DNA sequences account for 50–80% of the human genome 
. The repetition of DNA sequences causes ambiguities in alignment and genome assembly in DNA sequencing and poses a significant problem. This is magnified by next generation sequencing technologies as a result of the formation of shorter DNA fragments where fewer bases are present to verify the corresponding position in the reference genome 
. Uniform heterozygosity or excess heterozygosity resulting in Hardy Weinberg disequilibrium may suggest that a SNP has been identified as a result of sequence-read misalignment 
. Misalignment of sequenced DNA resulted in the erroneous identification of the heterozygote SNP rs13221869, which appeared to be in Hardy Weinberg equilibrium when genotyped using Sequenom in this study.
The steps necessary for the discovery of a new genetic variant ought to be threefold: firstly, the detection of the variant (often by large scale, high throughput approaches); secondly, the validation of this finding in an independent population and thirdly, characterization of the variant using an alternative technology 
. The National Center for Biotechnology Information CAV2
SNP genotype report lists 86 known SNPs in the CAV2
gene (accessed 06.11.2012). Of these, only 20 have been confirmed by population frequency or genotyping data, including two which were confirmed in populations of less than five individuals. This study identified six of the seven SNPs (rs8940, rs1052990, rs1055850, rs4727833, rs5886827, rs10249656, rs56213795) which have been validated in a population of European origin with a MAF>5% and provides population frequency data for an additional 54 SNPs. The Haploview plot of resequenced data illustrates the paucity of linkage disequilibrium between the confirmed SNPs (). For this reason, it is not feasible to accurately assess variation within the CAV2
gene using tag SNPs derived from the existing version of the HapMap project ().
At the turn of the millennium, the cost of sequencing the human genome was $100,000,000 
. Today, the human genome may be sequenced in its entirety for less than $1,000 
. This rapid reduction in the cost of DNA sequencing and the exponential increase in the output of sequencing platforms have resulted in an unprecedented amount of information about the genetic code becoming available. This, however, must be matched by accurate and reproducible bioinformatics platforms for analysis and, even more importantly, careful interpretation of the results. This investigation of CAV2
illustrates the importance of replication and detailed validation of findings in clinical genetic research.
Analysis of the human genome has provided useful insights into the pathogenesis of chronic kidney disease and kidney transplant outcomes 
. These insights have resulted in advances which are beginning to be translated into clinical practice 
is a plausible candidate gene for association with kidney transplant survival because of its proximity to the CAV1
locus and its modulatory role in fibrosis and angiogenesis which are key pathological components of chronic transplant dysfunction 
. The advantage of employing different technologies in the investigation of CAV2
is emphasised by this study.
This study did not identify a significant association between single nucleotide polymorphisms in CAV2 and kidney transplant outcomes. However, it did identify novel variants, provide frequency data for known variants and provide a plausible explanation as to how a functional SNP might have been mistakenly identified and reported.