Targeted - Next Generation Sequence (t-NGS) analysis was performed using a panel of 141 genes associated to the main Immunodeficiencies (list available upon request). For each gene, we analyzed the coding regions, 50 bp in each of the intronic boundaries, the promoter, and the 3’UTR for a total target size of about 1 Mb. ). A total of 50 ng of gDNA was processed through the SureSelectQXT Target Enrichment system (Agilent Technologies, Santa Clara, CA, USA) for Illumina multiplexed sequencing. Sequencing reactions were carried out on the MiSeq instrument (Illumina, San Diego, CA, USA) using a PE 150 2 flow cell, running 16 samples for each sequencing run to obtain an average coverage of about 200 (>95% of the gene’s target nucleotides are covered at >100 reads, with mapping quality score (MQ > 30) reads); 96% of the analyzable target regions were covered by at least 50x.
Less...