This presentation details bioscentia's established whole exome sequencing (WES) workflow, highlighting its role as the backbone of their molecular genetics department. Christian Beetz, head of molecular genetics, explains their application-based approach, emphasizing high throughput and cost-effectiveness using Roche's HyperExome v1 and v2. The workflow, supported by Hamilton and Roche Avenio Edge automation and two NovaSeq 6000 instruments, processes over 23,000 samples monthly. Key aspects include balanced capture oligos covering disease-relevant regions, stringent automated quality control measures (including contamination and sample identity checks), and the strategic shift from panel sequencing to WES for virtual panels and even single-gene analysis. While acknowledging the potential of long-read whole genome sequencing as a future direction, HyperExome v2 remains bioscentia's current standard due to its efficiency and cost profile.
0:00 Introduction of Speaker: Christian Beetz, Head of Molecular Genetics at bioscentia, a large human genetics laboratory within the Sonic Healthcare network, is introduced to discuss their whole exome workflow.
0:58 WES as Backbone: Whole exome sequencing (WES) is identified as the core application in bioscentia's molecular genetics department, despite offering newer methods like long-read whole genome sequencing.
2:11 Evolution of Sequencing: The lab's portfolio spans cytogenetics to next-generation sequencing (NGS), with WES having replaced earlier panel sequencing approaches and now combining with long-read WGS for future applications.
3:11 Extensive Experience with Roche HyperExome: Bioscentia is a long-term Roche customer, having processed over 17,000 samples with HyperExome v1 and 6,500 with HyperExome v2, totaling over 23,000 samples.
4:14 Exome vs. Whole Genome for Commercial Labs: WES is preferred over whole genome sequencing (WGS) for commercial labs due to better turnaround times and cost-effectiveness, as it focuses on approximately 43 megabases (coding exons and known genes) compared to the 3 gigabases of the entire human genome.
5:01 Efficient Sequencing Platform: The lab utilizes two NovaSeq 6000 instruments, typically loading 40 exome samples per S1 flow cell to achieve a cost-effective throughput.
5:40 HyperExome Content & Balancing: Roche's HyperExome features balanced capture oligos (baits) designed to cover all known exons, with a strong emphasis on regions known to be involved in human disease, drawing from databases like HGMD and ClinVar, even including some intronic pathogenic variants.
8:43 Automated Workflow: The WES workflow is largely automated using two Hamilton NGS Star instruments for library preparation (batches of 40-48 samples) and hybridization, which is typically done overnight.
10:50 Hybridization Process: The HyperExome uses biotin-labeled baits (120 bp complementary target oligos) to fish for DNA regions of interest, followed by magnetic bead binding and stringent washes, achieving an on-target rate typically greater than 80%.
12:11 Roche Avenio Edge Automation: The new Avenio Edge instrument, currently under evaluation, offers a highly automated, hands-off solution for the entire workflow (DNA to ready-to-load libraries), including pooling, quantification, and hybridization, significantly improving efficiency and requiring less specialized staff.
15:08 Comprehensive Quality Control (QC): An in-house bioinformatics team developed automated QC tools, checking mean coverage (aiming for 100x, with a minimum of 15x for variant calling), sample distribution in pools, regions with at least 15x coverage, and on-target rates (above 75%).
15:53 Sample Integrity Checks: QC includes verifying gender, adapter indexing, and contamination checks (identifying additional peaks in variant allele frequency distribution).
16:49 Sample Identity Verification: A rigorous identity check involves a second DNA preparation run with 24 SNPs (using Casp PCR) and comparing results to NGS data; at least 20 out of 24 SNPs must match to prevent sample mix-ups.
18:00 Exome as a Versatile Backbone: WES has fully replaced previous panel or single-gene analyses, serving as a "backbone" for creating virtual panels bioinformatically. Its streamlined approach makes it efficient even for analyzing a handful of variants.
19:13 Copy Number Variant (CNV) Detection: The even distribution of coverage allows for reliable CNV analysis, capable of detecting single-exon deletions (though single exons are confirmed by MLPA, while three consecutive exons are called with high confidence).
21:04 HyperExome v2 Improvements: Version 2 of the HyperExome has significantly improved coverage in previously missed exons (e.g., CNGN1, NF1), incorporating bioscentia's feedback.
21:43 Cost-Effective Mitochondrial Genome Coverage: By adding a small amount of non-enriched library to the enriched pool, the mitochondrial genome is completely covered at 30-50x depth without needing custom designs or extra analysis.
25:57 Future Outlook: While long-read whole genome sequencing offers access to previously uncovered regions, its current cost (4-5 times higher than exome) makes WES (HyperExome v2) the standard for over 80% of bioscentia's molecular genetics samples.
27:08 Q&A - Coverage Target: The lab aims for 70-100x mean coverage to ensure at least 15x coverage in all regions, which is considered the minimum for accurate heterozygous/homozygous variant calling, especially for challenging regions like the X chromosome in males.
28:07 Q&A - Avenio Edge Advantage: The primary advantage of the Avenio Edge is its complete hands-off automation, allowing samples to be loaded and a ready-to-load mix for sequencing to be retrieved, requiring less experienced staff.