Nimblegen seqcap ez human exome kit

Whole-exome sequencing was performed for validation of certain samples, listed in Supplementary Table 4. Using the Illumina Hiseq 4000, sequencing was conducted on genomic DNA: 1 μg of genomic DNA was fragmented using sonication (Covaris, S220), then optimized to give a distribution of 200–500 bp that was verified using a 2100 Bioanalyzer (Agilent, no. G2939BA). Library preparation was carried out using the Kapa DNA HTP Library Preparation Kit (KAPA Biosystems, no. 07 138 008 001). Hybridization of adapter-ligated DNA was performed at 47 °C for 64–72 h, to a biotin-labeled probe included in the Nimblegen SeqCap EZ Human Exome Kit (Roche, no. 06465692001). Libraries were sequenced using the Illumina Hiseq 4000 sequencing system and paired-end, 150-bp reads were generated for analysis with 200x coverage per sample. Exome data were analyzed using GATK v.3.7 with the human_g1k_v37_decoy as reference genome. Annotation of variants was performed using annovar (v.10-24-2019) and in-house pipelines.

Using the Illumina Hiseq 2500 sequencer, WES was conducted on genomic DNA extracted from 27 APAs along with paired adjacent normal adrenal and three APAs of known genotype (as sensitivity controls). GDNA (1 μg) was fragmented using sonication (Covaris, no. S220), optimized to give a distribution of 200–500 bp that was verified using a 2100 Bioanalyzer (Agilent, no. G2939BA). Library preparation was carried out using a Kapa DNA HTP Library Preparation Kit (KAPA Biosystems, no. 07 138 008 001). Hybridization of adapter-ligated DNA was performed at 47 °C for 64–72 h, to a biotin-labeled probe included in the Nimblegen SeqCap EZ Human Exome Kit (Roche, no. 06465692001). Libraries were sequenced using the Illumina Hiseq 2500 sequencing system, and paired-end, 101-bp reads were generated for analysis with 100x coverage per sample.