Adaptation of custom capture sequencing panels to the Oxford Nanopore MinION platform.

BACKGROUND

Next generation sequencing (NGS) remains underutilized in clinical microbiology applications despite providing broad pathogen spectrum detection superior to other molecular methods. This is primarily because of lower sensitivity of metagenomic NGS (mNGS) compared to PCR, lengthy turn-around times, cost, and complexity of data analysis. Capture sequencing is a technique that can mitigate some of the limitations of mNGS. Using probes that are engineered to selectively bind and pull down desired nucleic acids, capture sequencing enriches for targets of interest and can result in up to a 10,000-fold increase in sensitivity compared to mNGS. In this study, we describe the application of capture sequencing on Oxford Nanopore Technology's portable sequencer, the MinION MK1C.

METHODS

We examined the performance of VirCapSeq-VERT and TBDCapSeq, two distinct capture sequencing assays that target vertebrate viruses and tick-borne pathogens, respectively. Both assays were originally established on the Illumina platform. To enable sequencing on the MinION instrument, we developed a modified hybrid workflow using our established library preparation and capture protocol for Illumina, followed by the addition of the ONT sequencing adaptor. In tests using contrived and clinical samples, we compared sensitivity thresholds and sequencing output, including pathogen genome coverage and relevant read counts.

RESULTS

The addition of capture enrichment to MinION NGS provided significant improvement in pathogen detection when compared to mNGS. Assessment of assay performance on pathogen-positive samples revealed equivalent sensitivity on the MinION MK1C and Illumina NextSeq. We found that the elevated read counts and sequencing depth generated by Illumina NGS were offset by the greater read length obtained on the MinION MK1C and resulted in comparable pathogen genome coverage between the two platforms.

CONCLUSION

This study demonstrates the utility for employment of VirCapSeq and TBDCapSeq on different sequencing platforms and suggest the potential of the MinION platform for broad-spectrum clinical diagnostics.