High-throughput Nex- Gen clinical sequencing and reporting pipeline

Advancement of NGS applications in the clinical diagnostic arena requires methods for rapid delivery of high-quality sequencing data covering clinically relevant genes and HIPPA-compliant cloud-based tools for sample... [ view full abstract ]

Advancement of NGS applications in the clinical diagnostic arena requires methods for rapid delivery of high-quality sequencing data covering clinically relevant genes and HIPPA-compliant cloud-based tools for sample accessioning and reporting along with robust analysis pipelines for clinical applications. These combined methods enable rapid data generation, variant annotation, diagnostic interpretation and generation of clinical reports for large studies within a CAP/CLIA environment.

Our pipeline utilizes a ‘lightning capture’ process to deliver variant calls in 5-7 days after sample intake. The ‘lighting capture’ process includes: quick library preparation and capture enrichment, rapid sequencing and data analysis via Neptune, the HGSC-developed clinical pipeline. Methods employ a cost-effective 47plex co-capture format followed by sequencing 94 samples per lane for data generation. To ensure integrity of the end-to-end process, we have implemented the Fluidigm SNPTrace assay to confirm sample identity.

Application of this pipeline includes a capture panel (eMERGE-Seq, 535kb) of 109 genes and ~1500 unique single nucleotide variants of known clinical relevance for the National Institutes of Health eMERGE network. The design and process have been validated utilizing 647 control samples. High enrichment efficiency (>79% reads on target/buffer) and superior coverage metrics (99.7% of target bases ≥20x coverage, 339x average coverage) were observed. Initially 1332 bps with low coverage using the criteria of ≥10% of samples with coverage <20X was significantly reduced to 475 bps by utilizing a spike-in reagent supplemented into eMERGE-Seq. Detailed analysis using 42 de-identified samples found that known mutations were correctly identified with high confidence (100%), including CNV mutations. This pipeline, including secure cloud-based sample accessioning and automated clinical reports for review will be used to process >14,500 samples for the eMERGE program. To date, ~6000 eMERGE samples have been successfully sequenced in this clinical pipeline.