Sureselect human all exon v6 utr

To capture exonic DNA from formalin-fixed paraffin embedded tumor tissue and matched blood samples, we used SureSelect Human All Exon V6+UTR (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. The 101 bp–sequencing reads were aligned to the hg19 reference and further processed using the Genome Analysis ToolKit (GATK, v4.1.7.0) [20 (link)] in accordance with the best practices pipeline [21 (link)]. For RNA-seq, we used the SureSelectXT RNA Direct Reagent Kit (Agilent, USA) to construct sequencing libraries. The pre-processing of RNA-sequencing data were mapped to the GRCh38 human reference and conducted according to the National Cancer Institute (NCI) Genomic Data Commons workflow [22 (link)]. For quality control of sequencing, we performed qualimap (v2.2.1) with the aligned bam files [23 (link)].

Genomic DNA of peripheral blood leukocytes was extracted from NIID patients, and GGC repeat expansion in NOTCH2NLC was assessed by repeat primed PCR, and the number of GGC repeat expansions was confirmed with fluorescence amplicon length analysis as described in detail in a previous report [31 (link)]. In Cases 3 and 4, whole exome sequencing (WES) by NovaSeq 6000 and Agilent Sure Select Human All Exon V6 + UTR was performed, and data were analysed using 329 retinal disease-causing genes registered in Retinal Information Network (RetNet database [27 ]) to screen the pathogenic variants that can cause the retinal degeneration. We performed a visual inspection of pathogenic variant sites using the Integrative Genomics Viewer (IGV). The methods have been described in detail [15 (link)]. About ATXN7, one of the RetNet gene and the cause of spinocerebellar ataxia type 7 (SCA7), we investigated CAG repeat sequence expansion using a reported PCR method [14 (link)].