Fig. 1

Schematic representation of the next-generation sequencing data analytical workflow. After comparison with the reference sequence (GRCh38), 822 alleles different from the reference sequence (GRCh38) in patients with gout and non-gout controls were recorded (376 synonymous alleles and 160 nonsynonymous alleles in protein-coding genes, 52 alleles in tRNA genes, 61 alleles in ribosomal RNA (rRNA) genes, and 173 alleles in the noncoding region). In the Step 1, the enrichment of alleles in patients with gout and non-gout controls were identified using the chi-square test or exact test, as described in “Methods”. Next, gene burden ratio was calculated to characterize alleles as positively associated alleles or negatively associated alleles for Sequence Kernel Association Test (SKAT) analysis (Step 2). Eight genes associated with gout were obtained from SKAT analysis and annotated with Polymorphism Phenotyping (Polyphen), Sorting Intolerant From Tolerant (SIFT), Predict the pathology of MUTation (PMUT) (for alleles in protein-coding genes) (Step 3a) and the Human Mitochondrial Genome Database (mtDB), Multiple Alignment using Fast Fourier Transform (MAFFT), and Mammalian Mitochondrial tRNA Genes (Mamit-tRNA) (for alleles in transfer RNA (tRNA) genes) (Step 3b). Genotyping results of selected alleles was further validated (Step 4). After functional annotation, four alleles in protein-coding genes and four alleles in tRNA genes were singled out for genotype-phenotype correlations (Step 5)