Kaspar assays

Genomic DNA from the whole blood of patients with sarcoidosis was collected using the QIAamp DNA Blood Mini kit (QIAGEN). Genotyping of single nucleotide polymorphisms (SNPs; SNP database accession nos. rs2305619, rs3816527, and rs1840680) affecting the gene encoding PTX3 was performed using KASPar assays (LGC Biosearch Technologies) according to the manufacturer’s instructions in a 7500 Fast qPCR system (Applied Biosystems; Thermo Fisher Scientific). Genotyping sets included randomly selected replicates of previously typed samples. Agreement between original and duplicate samples was ≥99% for all SNPs. Laboratory personnel were blind to the sample status. Genotype frequencies of the selected SNPs were used to derive the haplotype configuration for each genotype. h1/h1, h1/h2, and h2/h2 haplotypes were defined as previously described (Cunha et al., 2014 (link)).

Whole-exome sequencing was performed on genomic DNA from patients and donors using QIAmp DNA Micro Kit (Qiagen). For whole-exome capture libraries, Agilent Human All Exon V7 baits were used. Samples were sequenced on Illumina NovaSeq 6000 (PE150) with mean coverage of 50× to generate 150 bp paired-end reads. After filtering using Trimmomatic v0.33 with default parameters (LEADING:15 TRAILING:15 SLIDINGWINDOW:4:15 MINLEN:50), reads were mapped against the GRCh38 reference genome using Burrows-Wheeler Aligner 3 (BWA-mem v0.7.17).³¹ (link) Duplicate reads were removed using Picard Tools (http://broadinstitute.github.io/picard/). Genome Analysis Toolkit 7³² (link) (GATK v4.2.4) was used for base quality recalibration and variant calling. The resulting variant call format (VCF) files for each sample were combined and genotyped using GATK CombineGVCFs and GenotypeGVCFs, respectively. For 15 of 18 SNPs with insufficient coverage, genotyping was performed using KASPar assays (LGC Biosearch Technologies).