After obtaining informed consent from all individuals recruited in the study, genomic DNA was isolated from their peripheral blood. The 9 F7 exons, their intronic boundaries, and untranslated regions of F7 gene were polymerase chain reaction (PCR)-amplified, followed by Sanger sequencing with the use of previously published primer sequences [2] (link). Sequence analyses were performed with Geneious Pro (version 10.0.6). Identified genomic variations were reported according to Human Genomic Variation Society nomenclature as presented in EAHAD F7 gene variant database (https://f7-db.eahad.org/ ).
Through PCR-Sanger sequencing, no causative mutation was identified in the entire F7 coding sequence of 3 out of 66 FVII-deficient patients. Whole-exome sequencing (WES) was therefore planned for those 3 cases. Exome enrichment was done by SureSelect library preparation kit (Agilent Technologies) and sequencing was performed on the HiSeq 4000 genome analyzer (Illumina), both at Macrogen The data were then analyzed as previously described [12 (link)]. Briefly, the reads were mapped to the human genome reference sequence (b37) using BWA v0.7.16 and further processed by Genome Analysis ToolKit v4.0.8 [13 (link)] and ANNOVAR [14 (link)]. Our in-house pipeline was used to filter and prioritize the variants.
Through PCR-Sanger sequencing, no causative mutation was identified in the entire F7 coding sequence of 3 out of 66 FVII-deficient patients. Whole-exome sequencing (WES) was therefore planned for those 3 cases. Exome enrichment was done by SureSelect library preparation kit (Agilent Technologies) and sequencing was performed on the HiSeq 4000 genome analyzer (Illumina), both at Macrogen The data were then analyzed as previously described [12 (link)]. Briefly, the reads were mapped to the human genome reference sequence (b37) using BWA v0.7.16 and further processed by Genome Analysis ToolKit v4.0.8 [13 (link)] and ANNOVAR [14 (link)]. Our in-house pipeline was used to filter and prioritize the variants.