DNA samples isolated from blood were prepared for exome sequencing using the Nextera Exome Enrichment method from Illumina, using 50 ng of gDNA. Whole exome sequencing samples were sequenced on Illumina HiSeq 2500 instruments. Reads were aligned to NCBI Build 37 of the human reference sequence using Burrows-Wheeler Aligner 0.7.8, and variants were identified using Genome Analysis Toolkit HaplotypeCaller 3.6 generating a genomic Variant Call Format. All positions with genotype quality score (GQ) > 19 and overall read depth (DP) > 7 were uploaded onto the RD-Connect Genome-Phenome Analysis Platform (https://platform.rd-connect.eu/genomics/ ). Data were initially analyzed applying standard filtering criteria (high/moderate variant effect predictor and minor allele frequency < 0.01) and a gene list of all known CM-causing genes. As no variants in known CM genes were identified, the full genome was interrogated in search of a novel candidate gene. Candidate variants were confirmed and segregated in unaffected family members by Sanger sequencing. To identify additional cases, a large cohort of more than 3,000 exomes was interrogated for homozygous or compound heterozygous rare variants in MACF1.
Nextera exome enrichment
Manufactured by Illumina
Sourced in Germany
The Nextera Exome enrichment is a library preparation kit designed for targeted sequencing of the human exome. It utilizes Illumina's proprietary Nextera technology to generate sequencing libraries from genomic DNA samples. The core function of this product is to selectively enrich for protein-coding regions of the genome, known as the exome, prior to sequencing.
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Lab products found in correlation
3 protocols using nextera exome enrichment
1
Exome Sequencing for Novel Gene Discovery
2
Comprehensive Genomic Profiling of TP53 Mutations
3
TP53 Mutation and Copy Number Analysis
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TP53 mutational status was assessed using whole-exome sequencing (WES, n = 90) or Sanger sequencing of exons 5 to 8 (n = 5). WES data were generated using Illumina Nextera Exome enrichment (n = 89) or TWIST Human Core Exome kit (n = 1) and sequenced on an Illumina NovaSeq within the Newcastle University Genomics Core Facility or Illumina HiSeq by Eurofins Genomics (Germany). Data were analysed using the Genome Analysis Toolkit (GATK 3.7) and variants called using Mutect2. PCR products for Sanger sequencing were amplified using primers designed for TP53 (Supplementary Table 1 ) and sequenced by Eurofins Genomics. WES base calls were confirmed by Sanger sequencing in 39 cases, with 100% concordance between sequencing methods.
Copy number alterations (CNAs) of 17p and other chromosomes were identified using Affymetrix Cytoscan HD, Genome-wide Human SNP Array 6.0 or OncoScan arrays performed by Eurofins Genomics. Raw data were analysed and visualised in Nexus Copy Number 10.0 (BioDiscovery) to detect CNAs and copy number neutral loss of heterozygosity (CNN-LOH) in all samples.
Copy number alterations (CNAs) of 17p and other chromosomes were identified using Affymetrix Cytoscan HD, Genome-wide Human SNP Array 6.0 or OncoScan arrays performed by Eurofins Genomics. Raw data were analysed and visualised in Nexus Copy Number 10.0 (BioDiscovery) to detect CNAs and copy number neutral loss of heterozygosity (CNN-LOH) in all samples.
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