NGS applications and workflows

Whole exome sequencing (WES) is a specific NGS application that provides coverage of ≥95% of the protein-coding (expressed) regions of the genome. Exonic and adjacent untranslated regions (UTR) comprise about 1% of the genome, but harbor the majority (85%) of disease-causing variants.⁵ WES is particularly well-suited for the detection of SNPs and indels, but is also capable of providing information on CNVs and structural variants. Although the WES sample preparation workflow (which requires enrichment with exome probes) is more complicated than for WGS, it offers significantly lower sequencing cost and a reduced analysis burden. WES is now routinely used in:

• Mendelian disease research
• Research of pediatric, rare, and complex diseases - particularly where single-gene or small panel investigations have failed to yield a possible genetic cause
• Oncology research

Exome probe sets have target sizes in the region of 30 - 60 Mb. Smaller exome panels may focus on actionable content, whereas larger target sizes typically include more adjacent, non-coding content to provide for the discovery of novel variants. The databases and algorithms used for probe design/selection are critical to ensure that all mutations of interest are adequately covered and minimize empirical optimization. 

Roche provides highly optimized, end-to-end solutions for exome sequencing using both manual and automated sample prep workflows.  

• Exome probes are based on the most recent genetic databases, expertly designed and selected, and empirically optimized for Roche sample preparation chemistries.
• Every lot of exome probes undergoes functional quality control (QC) testing to ensure quality and consistency.
• The automation-friendly KAPA HyperCap Evolved Workflow offers streamlined construction of high-quality libraries. An analytically validated WES sample preparation workflow for the AVENIO® Edge System offers walk-away automation. 
• High-quality libraries and superior probe design ensure excellent target enrichment metrics, high coverage uniformity, and low duplication rates; all of which contribute to sequencing efficiency and economy.⁴
• Easily integrated secondary and tertiary analysis and reporting, available via third-party collaborations and the Roche navify® Mutation Profiler, ensure reliable variant calls and comprehensible reporting.

Targeted sequencing generally refers to the analysis of defined and relatively small (<10 Mb) genomic regions of interest using next-generation sequencing (NGS). Reducing the targeted size allows for very deep coverage (precise analysis of low-frequency variants) and/or reduced per-sample sequencing cost. Targeted sequencing may be performed with pre-designed/analytically validated probe panels or assay, or with user-defined content. Both hybridization- and amplicon-based strategies are used to prepare and enrich libraries. Targeted sequencing is widely used in:

• Oncology research, particularly to track driver mutations
• Comprehensive genomic profiling (CGP) to analyze actionable germline or somatic variants
• Infectious disease analysis and surveillance
• Human Leukocyte Antigen (HLA) typing and other pharmacogenomic research applications

Content and probe performance are key to successful targeted sequencing. Probe sets must be designed to ensure that all desired variants are covered to the desired depth across all regions of interest, some of which may pose significant challenges to specificity and uniformity.

Roche offers a portfolio of solutions for versatile, high-confidence targeted sequencing.

• Our user-friendly, expert designer-assisted HyperDesign tool simplifies custom NGS panel design. With HyperDesign, you can create high-performing probe panels for almost any region of interest that does not require extensive empirical optimization⁴
• Catalog Design Share panels are pre-designed in collaboration with leading researchers for optimal coverage of relevant content in popular research areas
• Choose from the hybridization-based KAPA HyperCap Evolved Workflow or amplicon-based KAPA HyperPETE Workflow to achieve streamlined and robust sample prep that best suits your project scope, objectives, and operational constraints
• High molecule recovery with the KAPA EvoT4 Ligase, and high-fidelity, low-bias amplification with the KAPA EvoAmp ReadyMix mitigate against content distortion, even when preparing libraries from low inputs and challenging samples
• AVENIO Oncology Assay kits provide validated, easy-to-implement end-to-end solutions for in-house tumor profiling, CGP, surveillance, and monitoring

The transcriptome is composed of many different populations of RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA (such as microRNA and lncRNA). Unlike the genome, it can provide valuable insights into cellular behavior at a particular moment, whether at rest or when responding to changes in the environment, therapeutic treatments, or developmental progression. RNA sequencing (RNA-seq) is a powerful method for studying the whole transcriptome or selected parts in tissue or single cells, to:

• Identify genes that are differentially expressed in distinct cell populations and quantify their relative abundance
• Determine the effects of genetic variant splicing events
• Identify novel transcripts
• Detect gene fusions, isoforms, and other structural variants
• Identify single-nucleotide polymorphisms (SNPs)

A wide variety of RNA-seq workflows have been developed to support different experimental objectives and sample types (including available input and quality). To maximize the detection of low-abundance transcripts and reduce overall sequencing costs, many workflows incorporate an enrichment or depletion step to remove highly abundant, uninformative transcripts.

Roche supports RNA-seq workflows for whole transcriptome sequencing (WTS; analysis of coding- and non-coding transcripts) and mRNA-seq (analysis of coding transcripts only), with the KAPA RNA EvoPrep Kit and a choice of depletion/enrichment strategies (figure below). The KAPA RNA EvoPrep Kit offers streamlined and robust library preparation from a wide range of samples, including low-nanogram inputs and low-quality RNA from FFPE tissue.

NGS applications are always evolving as new and refined sample prep chemistries, sequencing technologies, and bioinformatics tools are introduced. Exciting emerging applications include:

• The use of artificial intelligence (AI) in NGS data analysis
• Multi-omics, focused on simultaneous analysis of DNA, RNA, proteins, and/or epigenetic signatures from bulk samples or single cells
• Quality control assays for CRISPR- and other gene or base editing technologies
• Spatial genomics/biology

Roche’s wide-range of sample preparation, sequencing, and analysis solutions are capable of preserving template molecules and minimizing experimental and analytical biases. This enables our customers to extract more reliable information from a wider range of inputs and sample types for clinical research, biotechnology, agricultural, and other applications—as evidenced from thousands of peer-reviewed publications citing our NGS products.⁴

Sample prep NGS applications that have recently been demonstrated or are currently being validated include:

• Robust analysis of liquid biopsy samples
• Compatibility of our exome sequencing chemistries with the Trinity™ and Cloudbreak Freestyle™ workflows on Element Biosciences™ AVITI™ sequencers
• Compatibility of KAPA EvoPrep with methylation conversion workflows for epigenetic profiling
• Comprehensive fusion detection from low-input FFPE tissue samples with KAPA HyperExome Probes V2
• An integrated workflow for exome sequencing from DNA and RNA
• Ultra-fast and efficient exome and targeted sequencing with the KAPA HyperCap Evolved Workflow for urgent samples