Purpose: The purpose of this study was to optimize the sequencing protocol for NoV detection on the Illumina MiSeq platform.
Methods: Norovirus-containing stool samples were suspended in 10% phosphate-buffered saline (suspension), centrifuged at 9,000 x g for 3 min (supernatant), and the supernatant was filtered through a 0.22 µM membrane filter (filtrate). Viral RNA was isolated from each of these three different preparations (suspension, supernatant and filtrate) with three different kits available in our lab: QIAamp viral RNA kit, RNAqueous kit and RNeasy mini kit. NoV RNA was quantified with real time RT-PCR and libraries were generated with same amount of RNA for each sample with and without oligo(dT) selection. Sequencing was performed on MiSeq and Genomic Workbench was employed for the data analysis.
Results: (1) The viral RNA yield with the RNAqueous kit was significantly (P < 0.05, n = 3) lower than from the other two kits; the highest viral RNA yield was obtained with the QIAamp viral RNA kit. (2) The filtrate, supernatant and suspension of the same stool sample showed similar sequencing outcomes, according to the total reads number, viral reads number, and percentage of viral specific reads. (3) The same mapping pattern - with more reads mapping towards the 3’-end, was shown for all oligo(dT) selected samples, but not for non-oligo(dT) selected samples.
Significance: These results show the importance of sample preparation optimization, RNA isolation and library generation for NGS using Illumina MiSeq for detection of Norovirus from stool samples.