Libraries were prepared with Nextera Rapid Capture Custom Enrichment Kit (Illumina Inc., San Diego, CA, USA), according to the manufacturer’s protocol. Preparation of libraries was performed starting from up to 10–150 ng of plasma ctDNA and 50–100 ng of gDNA from PBMCs (as corresponding normal reference, or hg19 when germ-line DNA from the patient was not available). gDNA was fragmented using transposones, adding simultaneously adapter sequences. For ctDNA libraries preparation was used NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England BioLabs Inc., Ipswich MA). Purified tagmented gDNA and ctDNA was used as template for subsequent PCR to introduce unique sample barcodes. Fragments’ size distribution of the DNA was assessed using the 2100 Bioanalyzer with a High Sensitivity DNA assay kit (Agilent Technologies, Santa Clara, CA). Equal amount of DNA libraries were pooled and subjected to targeted panel hybridization capture. Libraries were then sequenced using Illumina MiSeq or NextSeq500 sequencer (Illumina Inc., San Diego, CA, USA).
Nextera rapid capture custom enrichment kit
Manufactured by Illumina
Sourced in United States
The Nextera Rapid Capture Custom Enrichment Kit is a laboratory tool designed for targeted enrichment of genomic regions of interest. It enables the selective isolation and amplification of specific DNA sequences from a complex genomic sample.
Automatically generated - may contain errors
Lab products found in correlation
Nimblegen seqcap target enrichment, by Roche (2 mentions)
Nextera flex kit, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
High sensitivity dna assay kit, by Agilent Technologies (1 mentions)
Miniseq system, by Illumina (1 mentions)
Casava, by Illumina (1 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (1 mentions)
Puregene dna blood kit, by Qiagen (1 mentions)
Miseq personal sequencer, by Illumina (1 mentions)
10 protocols using nextera rapid capture custom enrichment kit
1
Plasma ctDNA Enrichment and Sequencing
2
Targeted NGS-based Genetic Profiling
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Following informed consent, patient genomic DNA was extracted from peripheral blood and, in two cases with EN (n. 17 and 26 in Table 1 ), also from the whole biopsy obtained from lesional skin. Mutations were identified through the targeted next generation sequencing (NGS) approach using a customized ichthyosis gene panel (Nextera Rapid Capture Custom Enrichment Kit, Illumina, San Diego, CA, USA; NimbleGen SeqCap Target Enrichment, Roche, Madison, WI, USA) and MiSeq or NextSeq550 sequencing platforms (Illumina). Identified variants were evaluated by the web-based tool VarSome [30 (link)] and validated by Sanger sequencing.
3
SARS-CoV-2 Whole-Exome Sequencing and Genetic Ancestry
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We obtained full exomes from DNA extracted from patients’ samples with SARS-CoV-2 infection. We used the Nextera Rapid Capture Custom Enrichment Kit or the Nextera Flex Kit (Illumina, San Diego, CA, USA) for library preparation and the IDT xgen-V1 kit for capture following manufacturer protocols. Whole-exome sequencing was performed on the NovaSeq 6000 equipment (Illumina, USA) with a 150-base paired-end dual index read format. Reads were aligned to the human reference GRCh38 using Burrow–Wheeler Aligner (BWA), algorithm MEM (https://github.com/lh3/bwa/tree/master/bwakit ). We also called genotypes using GATK HaplotypeCaller (version 4.0.9). We used the genotypes obtained in this step to infer the genetic ancestry. The pipeline used for alignment, variant calling, variant refinement, and genetic ancestry assessment is detailed elsewhere (22 (link)).
For the general elderly population (SABE), whole-genome sequencing was performed previously (31 (link)). Although SARS-Cov-2 infection is unknown for this cohort, this data provides a baseline for the frequency of each polymorphism in the general elderly population from São Paulo.
For the general elderly population (SABE), whole-genome sequencing was performed previously (31 (link)). Although SARS-Cov-2 infection is unknown for this cohort, this data provides a baseline for the frequency of each polymorphism in the general elderly population from São Paulo.
4
Comprehensive Cancer Gene Panel Sequencing
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A total of 503 MBC cases underwent next generation sequencing (NGS) of a custom panel of 50 cancer susceptibility genes including MUTYH. Briefly, paired-end libraries were prepared using the Nextera Rapid Capture Custom Enrichment kit (Illumina, San Diego, California, USA), pooled and loaded into a MiniSeq system (Illumina) for automated cluster generation, sequencing, and data analysis, including variant calling. Variant annotation and filtering was performed with Illumina Variant Studio Software version 2.2 against the human reference genome GRCh37. Variants were classified as pathogenic or likely pathogenic (collectively termed, pathogenic) according to the American College of Medical Genetics and Genomics (ACMG) recommendations (36 (link)). Briefly, variants were classified as pathogenic if they had a truncating, initiation codon or splice donor/acceptor effect or if pathogenicity was demonstrated by functional studies supportive of a damaging effect on the gene or gene product. All pathogenic variants were confirmed by double-stranded Sanger Sequencing (primer sequences are available upon request). Variants were named according to Human Genome Variation Society nomenclature (HGVS, http://www.hgvs.org ).
5
Curated Samples Sequencing Protocol
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Samples were curated and individuals were assessed as described previously [28 (link)]. Libraries were prepared for sequencing using Illumina Nextera Rapid Capture Custom Enrichment Kit (Cat ID FC-140-1009). The custom kit included 8,701 probes across the 2 Mb region for 288 samples (Project ID 44309). All samples were run on an Illumina HiSeq 2500 at 100 cycle pair end reads. Ninety-six multiplexed samples were run per flow cell with each multiplex being run twice on Rapid Run mode. Samples were de-multiplexed and converted to FASTQ files using Illumina software CASAVA.
6
Whole-Exome Sequencing with Nextera and IDT Kits
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We used the Nextera Rapid Capture Custom Enrichment Kit or the Nextera Flex Kit (Illumina, San Diego, CA, USA) for library preparation and the IDT xgen-V1 kit for capture following manufacturer protocols. Whole-exome sequencing was performed on the NovaSeq 6000 equipment (Illumina, USA) with a 150-base paired-end dual index read format. Reads were aligned to the human reference GRCh38 using Burrow–Wheeler Aligner (BWA) (https://github.com/lh3/bwa/tree/master/bwakit ). We also called genotypes using GATK HaplotypeCaller (version 4.0.9). The pipeline used for alignment, variant calling, variant refinement, and genetic ancestry assessment is detailed in the supplementary methods.
7
Customized Neuromuscular Disease Gene Panel
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To perform molecular analysis, we used a custom panel of 500 genes, including MTM1 and 88 other genes associated with neuromuscular diseases. We used the Nextera Rapid Capture Custom Enrichment Kit (Illumina, San Diego, CA) for library preparation and capture and the HiSeq2500 equipment (Illumina, San Diego, CA) for sequencing. The raw data (fastq files) was aligned against the GRCh37/hg19 human genome reference using BWA-MEM, with a postprocessing step using Picard Tools 1.81 to convert sam files to bam files and to mark PCR duplicates. We called genotypes using the Genome Analysis Toolkit (GATK) UnifiedGenotyper, version 3.7, following the GATK best practices. All variants were annotated using ANNOVAR.
8
Molecular Diagnosis of Inherited Ichthyosis
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All enrolled patients had received a molecular diagnosis on blood genomic DNA in our hospital. Mutation analysis of samples collected until 2018 was performed through the targeted NGS approach using a regularly updated customized ichthyosis gene panel (Nextera Rapid Capture Custom Enrichment Kit, Illumina, San Diego, CA, USA; NimbleGen SeqCap Target Enrichment, Roche, Madison, WI, USA) and MiSeq or NextSeq550 sequencing platforms (Illumina). More recently (2019â€"2021), mutations were identified through NGS analysis by Clinical Exome (Twist Bioscience, San Francisco, CA, USA), filtered for ARCI genes, and NovaSeq6000 sequencing platforms (Illumina). Identified variants were evaluated by the web-based tools VarSome [19] , Human Gene Mutation Database, and Leiden Open Variation Database. Variants were validated by Sanger sequencing in the patients and their parents. The mutations identified in 13 patients diagnosed till 2015 have been previously reported by our group [9] . Quantitative real-time PCR and/or microarray-based comparative genomic hybridization were employed to assess small genomic deletions or duplications (GRCh37).
9
Targeted Sequencing of Circulating Tumor DNA
10
Targeted NGS for Lipid Metabolism Genes
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Targeted NGS was performed using our LipidSeq panel, comprising 73 lipid metabolism-related genes including all specified non-LDLR FH-associated genes, namely, APOB, PCSK9, LDLRAP1, APOE, STAP1, LIPA, and ABCG5/8. Genomic DNA was isolated from whole blood using the Puregene DNA Blood Kit (Gentra Systems; Qiagen, Mississauga, ON). Libraries were prepared using the Nextera Rapid Capture Custom Enrichment Kit (Illumina, San Diego, CA), and enriched samples were sequenced on a MiSeq personal sequencer (Illumina) using 2 × 150 bp paired-end chemistry as described. A custom automated workflow in Genomics Workbench version 11.0.1 (CLC Bio, Aarhus, Denmark) was used for bioinformatic processing of raw sequencing data, generating .BAM (target region coverage data) and .VCF (variant calling data) files necessary for subsequent CNV analysis. Our LipidSeq method has an average depth of coverage (DOC) of 300-fold per base.
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