Seqcap ez vcrome 2

Manufactured by Roche

The SeqCap EZ VCRome 2.0 is a targeted enrichment solution for exome sequencing. It is designed to capture the coding regions of the human genome.

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Lab products found in correlation

Nextseq, by Illumina (4 mentions) Sureselect target enrichment system, by Agilent Technologies (4 mentions) Hiseq 2000, by Illumina (3 mentions) Idt xgen exome research panel v1, by Integrated DNA Technologies (3 mentions) Hiseq 2500 system, by Illumina (1 mentions) Sureselectxt target enrichment system, by Agilent Technologies (1 mentions) Novaseq, by Illumina (1 mentions) Hiseq platform, by Illumina (1 mentions) Agilent sureselect xt, by Agilent Technologies (1 mentions) Flow cell, by Illumina (1 mentions)

7 protocols using seqcap ez vcrome 2

Exome Sequencing Protocols for Genetic Analyses

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For P7, exomes were captured with the Agilent SureSelect Human All Exon V4+UTR kit (Agilent Technologies, Santa Clara, CA) and sequencing was performed on Illumina HiSeq 2000 machines using standard pair-end read sequencing protocol (Illumina, San Diego, CA). Analysis was as per Falk et al. 2014 and Li et al. 2014 . Possible pathological variants found were confirmed by Sanger sequencing.
Exome sequencing for P13 was performed clinically at the Baylor Whole Genome Lab. Briefly, exomes were captured using VCRome 2.1 in-solution capture, and sequenced on Illumina HiSeq using 100 bp paired-end reads. Data analysis and interpretation was as per Yang et al. 2013 . Possible pathological variants found were confirmed by Sanger sequencing.
Exome sequencing was performed in the affected individual P14 as well as in the nonaffected parents. Exomes were enriched in solution with SureSelect XT Target Enrichment System (Agilent Technologies) or SeqCap EZ VCRome 2.0 (Roche NimbleGen, Madison, WI) and sequenced as 100 bp paired-end runs on a HISeq2000 or HISeq 2500 system (Illumina).

Gil-Rodríguez M.C., Deardorff M.A., Ansari M., Tan C.A., Parenti I., Baquero-Montoya C., Ousager L.B., Puisac B., Hernández-Marcos M., Teresa-Rodrigo M.E., Marcos-Alcalde I., Wesselink J.J., Lusa-Bernal S., Bijlsma E.K., Braunholz D., Bueno-Martinez I., Clark D., Cooper N.S., Curry C.J., Fisher R., Fryer A., Ganesh J., Gervasini C., Gillessen-Kaesbach G., Guo Y., Hakonarson H., Hopkin R.J., Kaur M., Keating B.J., Kibaek M., Kinning E., Kleefstra T., Kline A.D., Kuchinskaya E., Larizza L., Li Y.R., Liu X., Mariani M., Picker J.D., Pié Á., Pozojevic J., Queralt E., Richer J., Roeder E., Sinha A., Scott R.H., So J., Wusik K.A., Wilson L., Zhang J., Gómez-Puertas P., Casale C.H., Ström L., Selicorni A., Ramos F.J., Jackson L.G., Krantz I.D., Das S., Hennekam R.C., Kaiser F.J., FitzPatrick D.R, & Pié J. (2015). De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes. Human mutation, 36(4).

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Targeted Sequencing Variant Filtering Protocol

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DES was performed as previously described (Farwell et al., 2015 (link); Farwell Hagman et al., 2017 (link)). In brief, samples were prepared using either the SureSelect Target Enrichment System (Agilent Technologies), SeqCap EZ VCRome 2.0 (Roche NimbleGen), or the IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies). Sequencing was performed using paired‐end, 100 or 150‐cycle chemistry on the Illumina HiSeq, NovaSeq, or NextSeq (Illumina). Bioinformatics filtering removed common benign variants, intergenic and 3′/5′‐untranslated region variants, intronic variants outside ±6, and nonsplice‐related synonymous variants. Family history‐based filtering and inheritance models were applied to the data. Alterations that have clinical overlap with the patient's reported phenotype and are classified as variants of uncertain significance or higher are reported (Farwell et al., 2015 (link)).

Towne M.C., Rossi M., Wayburn B., Huang J.M., Radtke K., Alcaraz W., Farwell Hagman K.D, & Shinde D.N. (2022). Diagnostic testing laboratories are valuable partners for disease gene discovery: 5‐year experience with GeneMatcher. Human Mutation, 43(6), 772-781.

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Exome Sequencing Workflow Comparison

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The exome sequencing was performed by two laboratories. Of all reports, 95 reports were analysed by Genome Diagnostics Nijmegen (Nijmegen, Netherlands), and nine reports were analysed by Ambry Genetics (Aliso Viejo, CA).
For the WES performed at Genome Diagnostics Nijmegen, the targets were enriched using Agilent SureSelectXT (Agilent Technologies), and the whole-exome sequencing was performed on an Illumina HiSeq platform (BGI, Copenhagen, Denmark), followed by data processing using BWA (read alignment) and GATK (variant calling). The variants were annotated using the external laboratory’s in-house-developed pipeline. The variants were prioritised using an in-house-designed ‘variant interface’ and manual curation.^{13 (link)} For the WES performed at Ambry Genetics, the samples were prepared using a SureSelect Target Enrichment System (Agilent Technologies) or SeqCap EZ VCRome 2.0 (Roche NimbleGen) and sequenced on an Illumina HiSeq 2000 or 2500. The initial data processing, base calling, alignments and variant calls were performed using various bioinformatics tools at Ambry Genetics. The variant calls were annotated using the Ambry Variant Analyzer tool (AVA)^{26 (link)} and filtered using laboratory-devised strategies.

Mak C.C., Leung G.K., Mok G.T., Yeung K.S., Yang W., Fung C.W., Chan S.H., Lee S.L., Lee N.C., Pfundt R., Lau Y.L, & Chung B.H. (2018). Exome sequencing for paediatric-onset diseases: impact of the extensive involvement of medical geneticists in the diagnostic odyssey. NPJ Genomic Medicine, 3, 19.

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Whole Exome Sequencing Protocol

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WES was performed at Ambry Genetics Corp. Genomic deoxyribonucleic acid was isolated from whole blood. Proband and parental samples were prepared using the SeqCap EZ VCRome 2.0 (Roche NimbleGen) or the IDT xGen Exome Research Panel V1.0. Final quantified libraries were seeded onto an Illumina flow cell and sequenced using paired-end, 100 or 150 cycle chemistry on the Illumina HiSeq or NextSeq. Variants were confirmed by Sanger sequencing.

Jacher J.E., Roy N., Ghaziuddin M, & Innis J.W. (2019). Expanding the phenotypic spectrum of MBOAT7-related intellectual disability. American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics, 180(7).

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Exome Sequencing and Bioinformatics Processing

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Exome sequencing and bioinformatics processing were performed as previously described [Farwell et al., 2015]. Briefly, patient samples were prepared using either the SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA), SeqCap EZ VCRome 2.0 (Roche NimbleGen, Mason, WI), or the IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies, Coralville, IA). Sequencing was performed using paired‐end, 100 or 150‐cycle chemistry on the Illumina HiSeq or NextSeq (Illumina, San Diego, CA). Bioinformatics filtering removed common benign variants, intergenic and 3′/5′ UTR variants, intronic variants outside ±2, and nonsplice‐related synonymous variants. Alterations that were previously classified as pathogenic or likely pathogenic and those that have an HGMD [Stenson et al., 2014] accession number were protected from filtering. Family history‐based filtering and inheritance models were applied to the data, and identified candidate alterations were subsequently confirmed using automated fluorescence dideoxy sequencing.

Smith E.D., Radtke K., Rossi M., Shinde D.N., Darabi S., El‐Khechen D., Powis Z., Helbig K., Waller K., Grange D.K., Tang S, & Farwell Hagman K.D. (2017). Classification of Genes: Standardized Clinical Validity Assessment of Gene–Disease Associations Aids Diagnostic Exome Analysis and Reclassifications. Human Mutation, 38(5), 600-608.

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Exome Sequencing Variant Identification

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Exome library preparation, sequencing, and bioinformatics were performed as previously described.^{15 (link)} Briefly, samples were prepared using either the SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA), SeqCap EZ VCRome 2.0 (Roche NimbleGen, Massion, WI),^{16 (link)} or the IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies, Coralville, IA) and sequenced using paired-end, 100- or 150-cycle chemistry on the Illumina HiSeq or NextSeq (Illumina, San Diego, CA). Stepwise filtering included the removal of common single-nucleotide polymorphisms, intergenic and 3′/5′ untranslated region variants, intronic variants outside ±2, and synonymous variants (other than potential splice-related synonymous changes at the first and last positions of exons). However, alterations classified as pathogenic or likely pathogenic based on Ambry's variant classification schema as well as alterations with a Human Gene Mutation Database identifier were protected from the aforementioned filtering. Identified candidate alterations were confirmed using automated fluorescence dideoxy sequencing.

Farwell Hagman K.D., Shinde D.N., Mroske C., Smith E., Radtke K., Shahmirzadi L., El-Khechen D., Powis Z., Chao E.C., Alcaraz W.A., Helbig K.L., Sajan S.A., Rossi M., Lu H.M., Huether R., Li S., Wu S., Nuñes M.E, & Tang S. (2016). Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases. Genetics in Medicine, 19(2), 224-235.

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Exome Sequencing Variant Analysis Pipeline

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Exome library preparation, sequencing, bioinformatics, and data analysis were performed as previously described [6 , 13 (link), 14 ]. Briefly, samples were prepared using SeqCap EZ VCRome 2.0 (Roche NimbleGen, Madison, WI) and sequenced using paired-end, 100-cycle chemistry on the Illumina HiSeq 2000 (Illumina, San Diego, CA). The sequence data were aligned to the reference human genome (GRCh37) and variant calls were generated using GATK and CASAVA. Variant filtering on WES was performed using a custom bioinformatics pipeline as previously described in detail [6 ]. Briefly, stepwise filtering included the removal of variants with quality scores <20 and allele counts <10X, common SNPs, intergenic and 3’/5’ UTR variants, non-splice-related intronic variants, and synonymous variants. Variants were filtered further based on family history and possible inheritance models. Data were annotated with the Ambry Variant Analyzer tool (AVA) [8 ]. All samples were required to meet minimum quality standards, with at least 90% of bases covered at ≥10X and having base call quality scores ≥Q20, which translates to a base-calling error rate of 1:100. Identified candidate alterations were confirmed using automated fluorescence dideoxy sequencing. All gene/bases covered in gene panel design were covered at >20X in the exome design.

LaDuca H., Farwell K.D., Vuong H., Lu H.M., Mu W., Shahmirzadi L., Tang S., Chen J., Bhide S, & Chao E.C. (2017). Exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels. PLoS ONE, 12(2), e0170843.

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