Custom AmpliSeq design for comprehensive, cost-effective genotyping of vitamin D associated genes in chronic kidney disease cohorts

Chronic kidney disease (CKD) is a major public health concern representing a significant financial burden to healthcare systems. Vitamin D levels are altered in individuals with CKD and several genetic variations in vitamin D related genes are associated with CKD. Targeted next generation sequencing offers unparalleled opportunities to efficiently and inexpensively examine customised genetic regions. Amplicons (n=329) were designed using AmpliSeq v2.2.1 (www.ampliseq.com). Resequencing was performed using two pools of 166 and 163 amplicons each, which covers 38 previously reported high value SNPs, coding + 50 bp flanking each exon, and untranslated regions for CYP2R1, CYP12B1, CYP24A1, CUBN, GC, NADSYN1, and VDR genes, which are important for vitamin D status. Following optimisation and validation, samples from two independent kidney disease cohorts from Belfast and Birmingham with extensive follow-up data were resequenced. Samples were prepared using Ampliseq kit 2.0 on an Ion Chef, or OneTouch2+ES machine, depending on which chip was being loaded. Reads were aligned using TorrentSuite v4 and SNP genotypes called using Partek Genomics Suite v6.6. The maximum number of alignments per sample was 118,222, with 261 SNPs identified, of which five were novel and 16 led to amino acid changes. We have developed a customised panel that enables cost-effective typing of large numbers of individuals (64 per 318 chip; 400 per P1 chip with >95% coverage at ≥30x) for 43.8 kb across vitamin D genes using 10 ng of DNA. Statistical association was examined between SNPs, serum total 25-OH vitamin D levels, and kidney function.