Nextera rapid capture exome

Exome sequencing libraries were generated using the Nextera Rapid Capture Exome (Illumina, San Diego, CA). Fifty ng of gDNA was used to generate a whole genome-sequencing library. Sequence libraries were subsequently pooled into sets of 12 prior to hybridization and enrichment. Note that all hybridizations, washes and enrichments were performed using the manufacturer’s recommendation. In all cases, libraries at both the whole genome and enriched exome stages were qualified and quantified using the DNA High sensitivity assay (BioAnalyzer 2100 Agilent). Exome sequencing was performed using the Illumina 2500 HiSEQ platform. Cluster generation and sequencing was completed using TruSeq PE Cluster Kit v3-cBot-HS and TruSeq SBS Kit v3-HS (200 cycle) kits respectively with libraries clustered at 8pM using the cBOT instrument (Illumina). Three sequencing reads were generated including 101bp paired-end sequencing reads and a single index read which allows for de-multiplexing of the library pools. Clustering and sequencing were performed following the manufacturer’s recommendations.

The DNA was extracted as described by Sambrook and collaborators [31 ]. The genetic material was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., USA). The libraries were prepared using the Nextera Rapid Capture Exome (Illumina) and SureSelect Human All Exon V6 (Agilent) Kits. Sequencing reactions were run using the NextSeq 500 High-output v2 300 Cycle Kit (Illumina®, USA) on the NextSeq 500® platform (Illumina®, USA).

DNA was extracted from dried blood spot in filter cards (Centocard)® using standard, spin column-based method. Approximately 37 Mb (214,405 exons) of the Consensus Coding Sequences (CCS) were enriched from fragmented genomic DNA by >340,000 probes that were designed against the human genome (Nextera Rapid Capture Exome, Illumina), and the generated library was sequenced on an Illumina NextSeq or HiSeq 4000 platform (Illumina) to an average coverage depth of 70–100X. An end-to-end inhouse bioinformatics pipeline, including base calling, primary filtering of low-quality reads and probable artifacts, and annotation of variants, was applied. All of the disease-causing variants that had been reported in HGMD®, ClinVar (class 1) as well as all variants with a minor allele frequency (MAF) of <1% in the ExAc database, were considered for this study. Our evaluation was focused on exons with intron boundaries ±20. All of the relevant inheritance patterns were considered, and family history and clinical information that were provided were used to evaluate the variants that we identified. Only the variants that were related to the phenotype are reported.

Whole exome sequencing was performed in an accredited commercial lab (Centogene-Germany) as follows. Approximately 37 Mb (214,405 exons) of the Consensus Coding Sequences were enriched from fragmented genomic DNA by >340,000 probes designed against the human genome (Nextera^®Rapid Capture Exome, Illumina), and the generated library was sequenced on an Illumina platform to an average coverage depth of 100–130×. End-to-end in-house bioinformatics pipelines, including base calling, primary filtering of low-quality reads and probable artifacts, and annotation variants were applied. All disease-causing variants reported in HGMD^®, in ClinVar, or in CentoMD^®, in addition to all variants with minor allele frequency (MAF) < 1% in the ExAc database were considered. Evaluation was focused on coding exons along with flanking ±20 intronic bases.

The DNA was extracted using the phenol-chloroform method described by Sambrook et al. [20 ]. The genetic material was quantified using a Nanodrop-8000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and the integrity of the DNA was assessed by electrophoresis in 2% agarose gel.
The libraries were prepared using the Nextera Rapid Capture Exome (Illumina) and SureSelect Human All Exon V6 (Agilent) kits following the manufacturer’s recommendations. The sequencing reactions were run in the NextSeq 500^® platform (Illumina^®, US) using the NextSeq 500 High-output v2 300 cycle kit (Illumina^®).

Whole-exome sequencing was performed for Patient II-8 as there were no DNA samples from the deceased siblings, patients II-1 and II-2. Approximately, 37 Mb (214,405 exons) of the consensus coding sequences were enriched from fragmented genomic DNA by >340,000 probes that were designed against the human genome (Nextera Rapid Capture Exome, Illumina, CA, United States). The generated library was sequenced on an Illumina NextSeq or platform (Illumina) to an average coverage depth of 70–100 ×. An end-to-end, in-house bioinformatics pipeline, including base calling, primary filtering of low-quality reads, probable artifacts, and annotation of variants, was applied.
All the disease-causing variants reported in HGMD^®, ClinVar and all variants with a minor allele frequency of less than 1% in the ExAc database were considered for this study. Our evaluation focused on the exons with intron boundaries of ±20. All relevant inheritance patterns were considered, and the family history and clinical information were used to evaluate the identified variants. Only variants related to the phenotype have been reported.

Gene sequencing was performed mainly at the Beijing Kangso Medical Inspection, China. Genomic DNA was isolated from the peripheral blood samples of the trios using a DNA Midi Kit (Qiagen GmbH, Hilden, Germany). Custom-design in-solution hybridization (Agilent SureSelect, Agilent) was used to select panel genes. All known protein-encoding regions were captured and enriched. Whole-exome sequencing (WES) was performed on the Illumina HiSeq2500 and HiSeq4000 systems to an average read depth 100× covering 99.9% per sample. For WES enrichment, the Nextera Rapid Capture Exome (v1.2, Illumina, San Diego, CA, USA) was used. HiSeq reads were mapped to the human hg19 reference genome using the Burrows–Wheeler Aligner software. The Genome Analysis Toolkit (GATK) was used to detect duplicated reads and single-nucleotide variants. Patients and their relatives with non-conclusive genetic workup were followed up for further examination. Data were processed preliminarily according to the standard procedures of WES (8 (link)). Segregation analysis of the variants was performed in the available family members using Sanger sequencing. Repeat-primed PCR (RP-PCR) and GC-rich PCR (GC-PCR) were used for detecting CGG repeat expansions in GIPC1.