Use of Oxford Nanopore MinION to generate full-length sequences of the Blastocystis small subunit (SSU) rRNA gene

Authors: Maloney, Jenny; Molokin, Aleksey; Santin-Duran, Monica

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2020
Publication Date: 11/25/2020
Citation: Maloney, J.G., Molokin, A., Santin, M. 2020. Use of Oxford Nanopore MinION to generate full-length sequences of the Blastocystis small subunit (SSU) rRNA gene. Parasites & Vectors. 13(1):595. https://doi.org/10.1186/s13071-020-04484-6.
DOI: https://doi.org/10.1186/s13071-020-04484-6

Interpretive Summary: Blastocystis is one of the most common enteric parasites of humans and animals worldwide. Transmission is fecal-oral through ingestion of cyst-contaminated water or food. It is well recognized a remarkable genetic diversity in the SSU rRNA gene which is currently the only gene used for defining Blastocystis subtypes (STs). Yet, full-length reference sequences of this gene are available for only 16 subtypes of Blastocystis due in part to the technical difficulties associated with obtaining these sequences from complex samples. We have developed a method using Oxford Nanopore MinION long read sequencing and universal eukaryotic primers to produce full-length SSU rRNA gene sequences for Blastocystis. Seven Blastocystis samples representing five subtypes (ST1, ST4, ST10, ST11, and ST14) obtained both from cultured and feces were used for validation. We demonstrate that this method can be used to produce highly accurate full-length sequences from both cultured and fecal DNA isolates from all five subtypes including ST11 for which no full-length reference sequence currently exists and for an isolated that contained mixed ST10/ST14. The ability to produce full-length SSU rRNA gene sequences will facilitate studies to understand the role of genetic diversity in important aspects of Blastocystis biology such as transmission, host specificity, and pathogenicity. This information should be useful to other scientists, physician, veterinarians, and public health agencies working on Blastocystis.

Technical Abstract: Background: Blastocystis is one of the most common enteric parasites of humans and animals worldwide. It is well recognized that this ubiquitous protist displays a remarkable degree of genetic diversity in the SSU rRNA gene which is currently the only gene used for defining Blastocystis subtypes. Yet, full-length reference sequences of this gene are available for only 16 subtypes of Blastocystis due in part to the technical difficulties associated with obtaining these sequences from complex samples. Methods: We have developed a method using Oxford Nanopore MinION long read sequencing and universal eukaryotic primers to produce full-length (> 1700 bp) SSU rRNA gene sequences for Blastocystis. Seven Blastocystis specimens representing five subtypes (ST1, ST4, ST10, ST11, and ST14) obtained both from cultured and feces were used for validation. Results: We demonstrate that this method can be used to produce highly accurate full-length sequences from both cultured and fecal DNA isolates. Full-length sequences were successfully obtained from all five subtypes including ST11 for which no full-length reference sequence currently exists and for an isolated that contained mixed ST10/ST14. Conclusions: The suitability of the use of MinION long read sequencing technology to successfully generate full-length Blastocystis SSU rRNA gene sequences was demonstrated. The ability to produce full-length SSU rRNA gene sequences is key in understanding the role of genetic diversity in important aspects of Blastocystis biology such as transmission, host specificity, and pathogenicity.