Axygen dna mini kit

Whole−exome sequencing (WES) was conducted on samples from patients with suspected CAPS. Genomic DNA was extracted from whole blood using the Axygen DNA Mini Kit (Axygen, China). A minimum of 3 μg of DNA was used for the indexed Illumina libraries (MyGenostics, China). Sanger sequencing was performed by MyGenostics to verify the mutation. Deep sequencing was also performed for patients with suspected CAPS, but for whom no NLRP3 mutations were found by WES. HGMD and OMIM databases were searched for suspected variants. The pathogenicity of each variant was calculated according to ACMG criteria and the pathogenicity of the variants was speculated by Polyphen, SIFT, GERP++, and Mutation-Taster.

WGS was performed on 25 CRKO complex strains. Genomic DNA was extracted using the Axygen DNA Mini Kit (Axygen, America) and sequenced by the Illumina Miseq system (Illumina, CA, USA) using a paired-end library with an average insert size of 350 bp. The quality filtered reads were assembled de novo utilizing SPAdes v3.11 (Bankevich et al., 2012 (link)). Average nucleotide identity (ANI) analysis was used to define species. To identify the acquired antimicrobial resistance (AMR) genes, all isolate genomes were genotyped for resistance by ResFinder, a Center for Genomics Epidemiology (CGE) bioinformatics tool (Zankari et al., 2012 (link)). The MLST of CRKO complex strains were analyzed by PubMLST¹ (Jolley et al., 2018 (link)).

T cells, B cells, and monocytes from P4 were sorted for deep sequencing further. PBMCs were stained with anti-CD3, anti-CD19, anti-CD14, and anti-CD16 antibodies (all from BioLegend, USA). Sorted cells were extracted using the Axygen DNA Mini Kit (Axygen, China). DNA sequencing was conducted as described above. PBMCs were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD45 RA, anti-CD27, anti-CD19, anti-CD24, anti-IgD, anti-CD38, anti-CD16, anti-CD56, anti-β, and anti-γδ antibodies (all from BioLegend, USA).