Assessing RNA Integrity by Digital RT-PCR: Influence of Extraction, Storage, and Matrices

The development of high-throughput sequencing has greatly improved our knowledge of microbial diversity in aquatic environments and its evolution in highly diverse ecosystems. Relevant microbial diversity description based on high throughput sequencing relies on good quality of the nucleic acid recovered. Indeed, long genetic fragments are more informative for identifying mutation combinations that characterize variants or species in complex samples. This study describes a new analytical method based on digital PCR partitioning technology for assessing the fragmentation of nucleic acid and more specifically viral RNA. This method allows to overcome limits associated to hydrolysis probe-based assay by focusing on the distance between different amplicons, and not as usually on the size of amplicons. RNA integrity can thus be determined as a new fragmentation index, so called Fragment size 50. Application of this method has provided information on issues that are inerrant in environmental analyses, such as the storage impact of raw samples or extracted RNA, extraction methods or the nature of the sample on the integrity of viral RNA. Finally, the estimation of fragment size by dPCR showed a very strong similarity with the fragment size sequenced using Oxford Nanopore Technology. In addition to enabling objective improvements in analytical methods, this approach could become a systematic quality control prior to any long-read sequencing, avoiding insufficiently productive sequencing runs or biases in the representativeness of sequenced fragments.