Basespace pipeline

Library preparation and whole exome capture were performed by using the Twist Human Core Exome Kit (Twist Bioscience, South San Francisco, CA, USA) according to the manufacture’s protocol and sequenced on the Illumina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina, Inc., San Diego, CA, USA) and the TGex software (LifeMap Sciences, Inc., Alameda, CA, USA) were used for the variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. Based on the guidelines of the American College of Medical Genetics and Genomics, a minimum depth coverage of 30× was considered suitable for analysis. Variants were examined for coverage and Qscore (minimum threshold of 30) and visualized by the Integrative Genome Viewer (IGV).

Library preparation and whole exome capture were performed by using the Twist Human Core Exome Kit (Twist Bioscience) according to the manufacture's protocol and sequenced on the Illumina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina) and the TGex software (LifeMap Sciences) were used for the variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), a minimum depth coverage of 30X was considered suitable for analysis. Variants were examined for coverage and Qscore (minimum threshold of 30), and visualized by the Integrative Genome Viewer (IGV). For this study, we analyzed only data on the ACE2 candidate gene.

Genomic DNA was isolated from peripheral blood using the QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany) following manufacturer's instructions. Library preparation and clinical exome capture were performed using the Twist Custom Panel kit (Twist Bioscience, San Francisco, CA, USA) and sequenced on the NovaSeq 6000 platform (Illumina). The BaseSpace pipeline (Illumina) and the TGex software (LifeMap Sciences) were used for variant calling and annotation. Reads were aligned to human genome build GRCh37/hg19. Based on the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP), a minimum depth coverage of 20X was considered suitable for analysis. The mean coverage for the target region was 152X.

Genomic DNA was extracted from peripheral blood samples using standard procedures and the Qiagen blood DNA mini Kit (Qiagen, Hilden, Germany). Library pre-paration and whole exome capture were performed by using the Twist Human Core Exome Kit (Twist Bioscience, South San Francisco, CA, USA) according to the manufacture’s protocol and sequenced on the Illu- mina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina, Inc., San Diego, CA, USA) and the TGex software (LifeMap Sciences, Inc., Alameda, CA, USA) were used for the variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. A minimum depth coverage of 30X was considered suitable for analysis, based on the guidelines of the American College of Medical Genetics and Genomics. All variants were examined for coverage and Qscore (minimum threshold of 30) and visualized by the Integrative Genome Viewer.

Total RNA from the positive and negative populations was isolated using the TRIzol® LS reagent according to manufacturer’s protocol. Following isolation, the RNA samples were treated with Turbo DNase (Thermo Fisher Scientific), followed by re-extraction with the Tri-Reagent to remove DNase. RNA quality was assessed on a Bioanalyzer Nanochip (Agilent, USA), and only samples with RNA integrity number (RIN) ≥ 8.0 were considered for sequencing (all samples except POS7: 7.5 RIN). Sample library preparation for RNA sequencing was accomplished using the Illumina TruSeq RNA library protocol (mean insert size of 150 bp). The samples were sequenced on Illumina Nextseq 500 high output v2 platform (2 × 40 bp), which generated an average of 50 million paired-end reads per replicate. The Illumina BaseSpace pipeline was used for de-multiplexing and filtering high-quality sequencing reads. Additional quality filtering steps were performed using Trimmomatic v0.36 [47 (link)] to remove adapters, leading and trailing low-quality bases (below quality 3), very short reads (shorter than 20 bases), and reads with less than an average quality score of 20 using a sliding window of 4 bases. The quality of the preprocessed and the processed data was verified using FastQC [48 ].

WES was performed on all the members of the four families using the Twist Human Core Exome Kit (Twist Bioscience, San Francisco, United States) according to the manufacturer’s protocol and sequenced with the Illumina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina, San Diego, United States) and the TGex software (LifeMap Sciences, Alameda, United States) were used for the variant calling and annotation, respectively. Sequencing data were aligned to the GRCh37/hg19 human reference genome. Variants with a coverage lower than 10×, genotype quality (GQ) < 15, and gnomAD minor allele frequency (MAF) > 5% were excluded. WES results were interpreted according to ACMG guidelines 2015 (Richards et al., 2015 (link)).

Genomic DNA was extracted from peripheral blood samples using standard procedures and Qiagen blood DNA mini Kit (Qiagen, Hilden, Germany). Library preparation and whole exome capture were performed by using the Twist Human Core Exome Kit (Twist Bioscience, South San Francisco, CA, USA) according to the manufacture’s protocol and sequenced on the Illumina NovaSeq 6000 platform. The BaseSpace pipeline (Illumina, Inc., San Diego, CA, USA) and the TGex software (LifeMap Sciences, Inc., Alameda, CA, USA) were used for the variant calling and annotating variants, respectively. Sequencing data were aligned to the hg19 human reference genome. A minimum depth coverage of 30X was considered suitable for analysis, based on the guidelines of the American College of Medical Genetics and Genomics. All variants were examined for coverage and Qscore (minimum threshold of 30) and visualized by the Integrative Genome Viewer (IGV).