7900ht sequence detection system

Genome sequencing was performed on the DNA samples from individuals I-1, I-2 and II-1. Libraries were prepared with the DNA tagmentation based library preparation kit (Illumina) without PCR, with 500 ng gDNA input. Library preparation was followed by clean up and/or size selection using SPRI beads (Beckman Coulter Genomics). After library quantification (Qubit, Life Technologies) and validation (Agilent Tape Station), equimolar amounts of library were pooled. The library pools were quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System and then sequenced on an Illumina NovaSeq6000 sequencing instrument with a paired-end 2 × 150 bp protocol (Target coverage 300x or 1200 Gb per sample). Sequence reads were mapped to the genome version GRCh37 (UCSC hg19) with the Burrows-Wheeler Aligner (BWA MEM). Single-nucleotide variants and short indels were called with the Genome Analysis Toolkit (GATK) according to the GATK Best Practices (McKenna et al. 2010 (link); DePristo et al. 2011 (link)). We used Jannovar (Jäger et al. 2014 (link)) for variant annotation, and Varfish for filtering and further data analysis as described previously (Holtgrewe et al. 2020 (link)).

RNA-seq analysis was carried out on HeLa cells transfected with siRNAs targeting endogenous CWC22, eIF4A3 or the negative control luciferase 48 h prior to harvesting. Two biological replicates were analyzed for each sample. Total RNA was extracted with Isol-RNA lysis reagent (5 Prime) and 1 μg of RNA was used as input material for library preparation. Ribosomal depletion and strand specific library preparation was carried out with the TruSeq® Stranded Total RNA LT (with Ribo-Zero™ Human/Mouse/Rat) according to the manufacturer's instructions. After validation (Agilent 2200 TapeStation) and quantification (Invitrogen Qubit System) all six transcriptome libraries were pooled. The pool was quantified using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System and loaded on two runs of Illumina Miseq sequencers with a 2×75bp v3 protocol. The analysis produced 1.4–1.5 Gb/sample, corresponding to 8.8–9.8 M reads/sample. Basic read quality check was carried out using FastQC showing >94% of Q30 bases (PF).