Next-generation sequencing (NGS) technology is becoming a powerful tool in a public health setting for virus surveillance. Two of the most common sequencing platforms are the Illumina MiSeq and the ThermoFisher Ion Torrent PGM. Despite extensive use of these sequencing platforms worldwide, there is limited information detailing a direct comparison with respect to virus sequencing. A comprehensive comparative analysis with regard to quantity and quality of data, as well as a cost analysis per virus genome per sample is urgently needed.
A panel of sixteen samples with representative virus were selected from pilot experiments; these include specimens containing picornaviruses and caliciviruses of different titer, and co-infection of the two viruses. Out of the sixteen samples, twelve were clinical specimens (stool and nasopharyngeal swabs), and four were cell culture isolates. For each sample, viral RNA was converted to cDNA using sequence-independent single-primer amplification, followed by three types of library constructions - Illumina (NexteraXT and KAPA HyperPlus kits) and Ion Torrent (KAPA DNA Library Preparation Kit). Libraries were multiplexed using the same quantification method for each platform and sequenced using a 500v2 and a 500v2 Nano kit for the MiSeq platform and a 316 and a 318 semi-conductor chip for the Ion Torrent platform. The resulting sequencing data sets were analyzed by an in-house bioinformatics pipeline for viral genome assembly and general platform specific run data was collected and compared using Geneious v.9.1.6.
For this dataset, Illumina MiSeq sequencing generated more viral reads and had a greater average depth of coverage, with higher quality scores, than Ion Torrent. The Illumina MiSeq detected all low titer virus clinical specimens and co-infections while the Ion Torrent was unable to detect some of these specimens. For multiplexed libraries on the Ion Torrent PGM, the 318 chip was a better choice for depth of coverage and analytical sensitivity for viral detection and identification. For the library multiplexing used for this dataset, the Illumina MiSeq sequencing platform was the better choice for the cost per sample.
Overall, the normalized average depth of coverage was higher for the Illumina MiSeq 500v2 kit platform versus the Ion Torrent PGM 316 or 318 chips and the MiSeq 500v2 Nano kit. In this set of experiments, the cost per sample was $50 less on the MiSeq platform, giving Illumina a clear advantage in both cost and quality of sequencing for viruses.
Sequencing strategies and technology advancements using the various NGS platforms , Human, non-human, and infectious disease applications
