Nextseq 550 sequencing platform

Quantitative reverse-transcription real time polymerase chain reaction (qRT-PCR) for BoDV-1 [4 (link)] was conducted from CSF of seropositive patients and from available formalin-fixed paraffin-embedded (FFPE) brain tissues. For complete virus genome reconstruction, a diagnostic sample underwent unbiased next-generation sequencing (NGS) using a NextSeq550 Illumina sequencing platform as described elsewhere [13 ].
Molecular relationships of BoDV-1 sequences were analyzed by constructing a phylogenetic tree using the maximum likelihood method in PhyML 3.0 (https://www.atgc-montpellier.fr/phyml/versions.php) with 1000 pseudo-replicates based on sequences from the nucleoprotein to the phosphoprotein gene (1824 nt, representing genome positions 54–1877 of BoDV-1 reference genome U04608). For the assessment of specific node support, subtree pruning and regrafting (SPR), branch-swapping and an approximate likelihood ratio test (aLRT) were performed. The Akaike information criterion was chosen as the model selection framework and the GTR + I + G as the best model.

Karyotypes were determined by chromosomal G-banding. The results of the analysis for ETV6::RUNX1 translocation by FISH of the leukemia samples obtained during routine diagnostics were compiled from the patient records for this study (Suppl. Tables S2 and S3). To detect CN alterations and CN neutral loss of heterozygosity (CNN-LOH), all 60 leukemia samples were analyzed using SNP-arrays. For 18 leukemia cases, the CytoScan HD array (Thermo Fisher Scientific, Waltham, MA) was used as described.^{21 (link)} The data were initially aligned to GRCh37 and a lift-over to GRCh38 was performed for downstream analysis. The remaining 42 leukemia cases were analyzed using the Illumina CytoSNP-12 v2.1 array (Illumina, San Diego, CA) according to the manufacturer’s protocol. The array was scanned using the scanner option on the NextSeq550 sequencing platform (Illumina). The raw data were processed and analyzed using the Beeline 2.0.3.3 and the BlueFuse Multi 4.5 software from Illumina. The human reference genome GRCh38 was used. We included SVs encompassing ≥20 probes, ≥50 kb for deletions and duplications, or ≥5 Mb for CNN-LOH. For all samples, B- and T-cell receptor rearrangements were excluded from downstream analysis and SVs overlapping with centromere or reference gap regions. The filtered molecular genetic results are summarized in Suppl. Table S4.

Leaf samples were collected from five plants at the 4 week growth stage. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to manufacturer’s instructions and used to produce libraries using TruSeq RNA library Prep Kit v2 (Illumina). Pooled libraries were sequenced in a NextSeq550 sequencing platform (Illumina). Two biological replicates were generated for each genotype, and at least 20 million reads were produced per replicate.

The peripheral blood of the subjects was collected into vacutainer tube with EDTA. DNA was extracted using the phenol-chloroform method (Sambrook and Russell, 2006 ), quantified using Nanodrop fluorometer (Thermo Fisher) and integrity evaluated by electrophoresis in 2% agarose gel.
Whole-exome sequencing libraries were prepared using Nextera Rapid Capture Exome (Illumina) and SureSelect Human XT all exon V6 (Agilent) kits following the manufacturer recommendations. The libraries were sequenced in the NextSeq 550 sequencing platform (Illumina) in 4 NextSeq 500/550 High Output Kit runs with approximately 16 samples each.

Thirty libraries of paired-end short reads obtained with Illumina NextSeq550 sequencing platform were analyzed. Sequencing quality and length distributions were assessed using FastQC software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) version 0.11.5. Cutadapt software version 1.9.1 [38 ] was used to remove sequencing adapters, and Prinseq-lite software version 0.20.4 [39 (link)] was implemented for filtering with the following criteria: length not less than 100 bases, mean Phred quality score not less than 30. In case if one read in a pair did not meet the above criteria, the whole pair was discarded from analysis. Additionally, Prinseq option ‘-derep 1’ was employed in order to remove possible optical replicates.
Libraries of short reads were aligned to the reference genome version SolTub_3.0 taken from Ensembl plants database [29 (link)] using STAR aligner version 2.5.3a [40 (link)].

Two children affected with EBS-KLHL24 were studied (PT-1 and PT-2). PT-1 has been described (Case 1, (22 (link))), while PT-2 is a previously unreported 7-year-old female child, born to healthy non consanguineous healthy parents. Specifically, EBS-KLHL24 biopsies were taken from perilesional unblistered skin following skin rubbing, which results in basal keratinocyte vacuolization and microblistering, only detectable at microscopy analysis. Patient skin biopsies and blood samples were obtained after informed consent, with the approval of the Ethics Committees of participating Institutions and in conformity with the Helsinki guidelines. Normal human keratinocytes (NHKs) were obtained from skin or foreskin of age-matched healthy subjects undergoing surgery.
Skin biopsies were used for immunofluorescence antigen mapping and keratinocyte cultures as described (77 (link)). PT-2 genomic DNA was extracted from peripheral blood using QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany), and sequence variants were identified through Next Generation Sequencing (NGS) approach (NimbleGenSeqCap Target Enrichment—Roche, Madison, WI, USA; Twist Human Core Exome Kit—Twist Bioscience, San Francisco, CA, USA) and NextSeq550 sequencing platform (Illumina, San Diego, CA, USA). Variant validation and segregation analysis were performed by Sanger sequencing.