Ampure spri beads

Whole exome sequencing was performed for the two affected children (III-1, III-2) at the Beijing Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Exome sequences were enriched using an Agilent liquid capture system (Agilent SureSelect Human All Exon V6; Agilent Technologies, Santa Clara, CA, U.S.A.) according to the manufacturer’s protocol. First, genomic DNA was randomly fragmented to an average size of 180–280 bp using a Covaris S220 sonicator (Covaris, Brighton, U.K.). Second, the DNA fragments were end-repaired and phosphorylated, followed by A-tailing and ligation at the 3′ ends with paired-end adaptors (Illumina, San Diego, CA, U.S.A.) with a single ‘T’ base overhang, and purified using AMPure SPRI beads from Agencourt (Azincourt, France). Then, the size distribution and concentration of the libraries were determined using an Agilent 2100 Bioanalyzer and qualified by using real-time PCR. The DNA libraries were then sequenced on an IlluminaHiSeq 4000 sequencer for paired-end 150 bp reads at Beijing Novogene Bioinformatics Technology Co., Ltd. The raw data were saved as a FASTQ (fq) format file.

Two separate CRISPR primer (CRISPR1 and CRISPR2 locus) pairs were designed for two first rounds of PCR. Two microliters of template DNA entered a first round of PCR. The primer design incorporates a recognition sequence to allow a secondary nested PCR process. Samples were first purified with Ampure SPRI Beads before entering the second PCR performed to incorporate Illumina adapter sequences. Samples were purified using Ampure SPRI Beads before being quantified using Qubit and assessed for size distribution using the Fragment Analyzer (Agilent). Successfully generated amplicon libraries were taken forward and pooled in equimolar amounts, then size selected with a Pippin Prep machine (Sage Science) using a range of 180–600 bps. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa on a Roche Light Cycler LC480II according to the manufacturer’s instructions. Template DNA was denatured according to the protocol described in the Illumina cBot User guide (Illumina Document #15006165 v05) and loaded at 12.5 pM concentration. Fragmented PhiX phage genome was added to the sequence library at 15% in order to increase the sequence complexity. The sequencing of each pool was carried out on one lane of an Illumina MiSeq, at 2×250 bp paired-end sequencing with v2 chemistry.