PW02-026 - Low frequency variants of NLRP3 in CAPS patients

Methods All exons of NLRP3 were amplified by PCR (30 cycles) from genomic DNA isolated from PBMCs of healthy controls or CAPS patients. Thereafter, PCR products were concatenated, fragmented and subjected to NGS fragment library preparation followed by Illumina short read sequencing. For SNV calling a customized pipeline on basis of the GATK pipeline (1000 Genomes project) was utilized using a 40.000x coverage to assure sufficient sensitivity. In order to determine the accuracy of quantification, PCR products containing a known heterozygous mutation (T348M) were mixed with NLRP3 wildtype PCR products to obtain dilutions of the mutated sequences of 25%, 12.5%, and 6.25%.