Nextera rapid capture kit

Genomic DNA was isolated from whole blood of the proband and both parents. Exome capture and library preparation was performed using the Nextera Rapid Capture kit (Illumina, San Diego, CA). Captured libraries were sequenced on an Illumina HiSeq 2000 (2 ×100 nucleotides) to a depth such that a minimum 80% of targeted bases were sequenced to a read depth of 0020× or greater. Reads were aligned to the reference human genome (GRCh37) using BWA-MEM [16 (link)], and pedigree informed variant calling was performed using the Real Time Genomics (Hamilton, New Zealand) integrated analysis tool rtgFamily v3.6.2 [17 (link)]. All variants were annotated using SnpEff v4.2 [18 (link)], SnpEff GRCh37.72 database, dbSNP138, and dbNSFP v2.9. Rare variants (MAF <0.01) were identified and assessed as previously described for their potential to disrupt protein function under different inheritance models using a custom-built interpretation interface incorporating evidence including minor allele frequency, conservation, predicted pathogenicity, and disease association [19 (link)].

DNA samples from two affected individuals (III-8 and III-10) were submitted to whole-exome sequencing. The library was prepared with Nextera rapid capture kit (Illumina), sequence capture was performed with Illumina Exome enrichment kit (~62 Mb target size) and sequencing was performed using HiSeq 2500. Fastq files were aligned against reference GRCh37 with Burrows-Wheeler Aligner (BWA) (68 (link)), realignment of indel regions, discovery of variants and recalibration of base qualities were performed using GATK software (69 (link)) for the production of VCF files; the VCF was annotated by ANNOVAR software (70 (link)). Variant frequencies were compared with public variant databases: 1000 Genomes (71 ), National Heart, Lung, and Blood Institute Exome Sequencing Project (NHLBI-ESP) (72 ), Genome Aggregation Database (gnomAD) (73 ) and Online Archive of Brazilian Mutations (ABraOM) (74 ). Polyphen-2 (75 (link)), SIFT (76 (link)), Provean (77 (link)) and MutationTaster2 (78 (link)) were used for in silico damage prediction to the protein. Protein sequence alignment near the best candidate variant was performed by Clustal Omega alignment program (79 ).

Adapter-ligated indexed libraries were generated using the Illumina Nextera Rapid Capture kit (Illumina) from 50 ng of DNA as per manufacturer’s instructions. The libraries were quantified using a Qubit High Sensitivity dsDNA assay (Life Technologies). Five hundred nanograms of adapter-ligated barcoded DNA from each sample from each library were pooled into a capture pool of 12. Each capture pool was hybridised twice with enrichment probes for the exome. The fragment sizes of enriched libraries were assessed using a bioanalyser (Agilent Technologies, Folsom, CA, USA) and quantified using KAPA Library Quantification Kits (Kapa Biosystems, Wilmington, MA, USA).
Paired-end 125-bp sequencing runs were performed on an Illumina HiSeq 2500 instrument, aiming for a mean read depth coverage of 100 for the NA12878 dilution series.

DNA samples, from the two affected members of Family 1, underwent whole-exome sequencing at OmegaBioservices (Norcross, GA, USA). An Illumina Nextera Rapid Capture Kit^® (37Mb; San Diego, CA, USA) was used for library preparation, according to the manufacturer’s instructions. The library was then sequenced using the Illumina HiSeq 2500 sequencer using the pair-end 150bp run format. The sequencing data were processed using the Illumina DRAGEN Germline Pipeline v3.2.8.

Exome sequencing libraries were prepared by using a Nextera Rapid Capture Kit (Illumina, Calif, USA) based on the manufacturers’ protocol with slight modifications. The library concentration was quantified by a Qubit dsDNA Broad Range Assay Kit (Invitrogen, USA). Library size was measured by using a LabChip 3 K Hisense Kit (PerkinElmer, USA). Paired-end exome sequencing with 150 bp cycles was performed on a HiSeq 4000 (Illumina, Calif, USA), targeting an averaged depth of 100X.