Table 2 Recommendations in the processing and genomic testing of cancer specimens for mutational analysis and interpretation
Process step | Issue | Recommendation |
|---|---|---|
Sample input | -Some MPS applications require large amounts of input DNA | -Efficient and high-throughput extraction methods are recommended (automation of extraction is suggested for tracking large numbers of samples) |
| Â | Â | -Low elution volumes are also recommended to maximise DNA input |
| Â | Â | -Standardized fixation methods and optimised storage conditions of tissue blocks that maximise the quality and quality of DNA extracted should be employed |
Sample quality control | -FFPE-derived DNA is often fragmented, limiting the amount of useable material for MPS | -Integration of a quality-control step that assesses DNA integrity before sequencing |
| Â | Â | -Use of auxiliary testing methods for samples that fail suitability for MPS |
Sequencing platform | -MPS platforms can range widely in sequencing data output, processing times, running costs | -Currently, benchtop sequencers are best suited for diagnostic purposes because of ease of use, manageable data outputs, quicker processing times and lower running costs |
Sequencing panel/assay | -Mutational profiling using MPS can range from a small panel of genes to whole exome/genome scale sequencing | -A small to medium panel of genes is generally preferred as it targets valuable sequence coverage to clinically informative genes rather than genes of low clinical value |
Bioinformatics processing of sequencing data | -MPS can generate immense amounts of sequencing data | -Adequate data storage based on local or cloud-based systems |
| Â | -Raw sequencing data require multiple processing steps to generate variant calls | -Automated and integrated bioinformatics pipeline dedicated to generate variants |
Variant filtering | -System noise, technical artefacts and rare SNPs can make detection of somatic mutations difficult | -Rule-based filtering of variants should be applied to ensure that only high confidence variants are analysed |
| Â | -Variants called from FFPE-derived DNA often display sequencing artefacts | -All actionable mutations should be validated internally though replication or/and through orthogonal testing |
Interpretation | -Variants of unknown biological or clinical relevance can often be identified | -Information based on known variant prediction analysis or literature-based/database evidence can aid in the interpretation of variants |
| Â | Â | -Multidisciplinary discussions in the interpretation of variants allowing a comprehensive and efficient approach in clinical management |
Reporting | -The number of variants produced from MPS data make it difficult to decide what to report to a clinician | -A concise report that describes variants of most clinical applicability and that provides decision support should be produced |
| Â | Â | -Comprehensive details of other relevant variants can be included supplementary to the main report |
Workflow management | -MPS dramatically increases the number of samples tested | -Incorporation of automation and a LIMS to streamline processes and shorten turnaround times |
| Â | -Owing to multiple loci tested, multiple mutations have to be analysed | -Implementation of a variant management system to catalogue mutations |
