Dna extraction protocol

We studied 35,601 WGS data from whole-blood DNA in the Genomics England 100,000 Genomes Rare Disease Main Programme²⁸ . DNA was extracted using Qiagen DNA extraction protocols and following quality assurance and quantification 4.5 µg of DNA was submitted to Illumina Inc at their Great Chesterford centre. After sample quality control (QC) (details below) (Fig. 1a), 11,035 trios were included in this study.

Genomic DNA from fin clips purified using Qiagen DNA extraction protocol were used as template for a two-step barcoding PCR targeting the CRISPR locus as described by Gagnon et al.^{50 (link)}. Briefly, each sample was given a unique 6-mer barcode (Trueseq barcode primers) where after the barcoded dnd PCR products were mixed in equimolar ratios (oligos forward: 5′ tct ttc cct aca cga cgc tct tcc gat ctC TAC ATG CAT CAT TC CCA CCC CA 3′, reverse: 5′ tgg agt tca gac gtg tgc tct tcc gat ctA AG TTC CAC CAT TAC ACT GCT T 3′). We used the same PCR primers as described in the above HRM protocol. The final denatured sequencing library was prepared at a concentration of 8 pM and spiked with 5% denatured phiX and sequenced on the MiSeq using MiSeq Reagent Kit v3 (600 cycle format).