Abi 3730xl analyzer

All 490 samples were amplified at 17 SSR nuclear loci (see details in Supplementary Note 1 and Supplementary Table 1). PCR amplification was performed in a total volume of 20 μl comprising 11 μl of 2 × Taq PCR MasterMix, 0.3 μl of each primer, 1 μl of primer template (10–50 ng), and 7.4 μl double-distilled H₂O. All amplifications were conducted using a PTC-2000 thermal cycler (MJ Research) as follows: 5 min at 94°C, followed by 32 cycles at 94°C for 40 s, at the specific annealing temperature (Tm) for each marker (Supplementary Table 1), and at 72°C for 90 s, with a final extension at 72°C for 10 min. The final PCR products were sequenced using an ABI 3730 XL Analyzer (Applied Biosystems, Foster City, CA, United States) and analyzed with GENEMARKER v2.2.0 (Holland and Parson, 2011 (link)).

The 16S rRNA gene sequencing was identified via polymerase chain reaction (PCR) with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) [41 (link)]. The PCR product was sequenced by the Tsingke Company (Xian, PR China) using the dideoxy chain termination method with an ABI 3730XL Analyzer (Applied Biosystems), and the complete gene sequencing of the 16S rRNA was complied with SeqMan software (Lasergene). For the 16S rRNA gene sequences EzBioCloud’s Identify services were used to obtain sequence information. The 16S rRNA gene sequencings were aligned using ClustalW [42 (link)]; then, the phylogenetic trees—based on 16S rRNA by the MEGA 11 [43 (link)] software package using the neighbor-joining (NJ) [44 (link)], minimum-evolution, and maximum-likelihood (ML) methods followed by bootstrap analysis with 1000 bootstrap resamplings [45 (link)]—were reconstructed. Kimura’s two-parameter model [46 (link)] was used as the model for estimating genetic differences of nucleotide substitution. The phylogenomic tree was reconstructed based on the up-to-date bacterial core gene set (UBCG), according to the pipeline suggested by Na et al. [47 (link)].

DNA samples were extracted from peripheral blood using a QIAmp DNA Blood Mini Kit (Qiagen, Germany). Libraries were prepared using TruSight One kit (Illumina, USA) in Family 1 and SureSelect Human All exon V2 kit (Agilent Technologies, USA) in Family 2 following manufacturer’s instructions. Paired-end sequencing was performed on an Illumina Genome Analyzer II platform (Illumina, USA). Reads were mapped against the human reference genome hg19 using the BWA software and analyzed with the GATK Unified Genotyper v2.8. Amplicon-based deep sequencing of specific exons of the PLCG2 gene (RefSeq NM_002661.3) was performed as previously described to evaluate parental gene mosaicism [10 (link)]. For Sanger sequencing, specific exons of the PLCG2 gene were amplified by in house–designed PCR (primers listed in Supplementary Table S2), purified with Illustra ExoStar 1-Step kit (GE Healthcare, USA), bidirectional sequenced using ABI BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and run on an automated ABI 3730XL analyzer (Applied Biosystems, USA).

The H34wt and the mutants were sequenced with a ABI 3730xl Analyzer (Applied Biosystems, CA, USA) by using ABI BigDye™ Terminator v3.1 Cycle Sequencing Kits. GENETIX Ver. 8 (GENETIX, Tokyo, Japan) software was used for sequence analysis and deduction of amino acid sequences.

The #7TR and H34 clones were sequenced with an ABI 3730xl Analyzer (Applied Biosystems, Waltham, MA, USA) using ABI BigDye™ Terminator v3.1 Cycle Sequencing Kits. GENETIX Ver. 8 (GENETIX, Tokyo, Japan) software was used for sequence analysis and deduction of amino acid sequences.