Mind the gap: new full-length sequences of Blastocystis subtypes generated via Oxford Nanopore Minion sequencing allow for comparisons between full-length and partial sequences of the small subunit of the ribosomal RNA gene

Authors: Maloney, Jenny; Santin-Duran, Monica

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2021
Publication Date: 5/5/2021
Citation: Maloney, J.G., Santin, M. 2021. Mind the gap: new full-length sequences of Blastocystis subtypes generated via Oxford Nanopore Minion sequencing allow for comparisons between full-length and partial sequences of the small subunit of the ribosomal RNA gene. Microorganisms. https://doi.org/10.3390/microorganisms9050997.
DOI: https://doi.org/10.3390/microorganisms9050997

Interpretive Summary: Blastocystis is a common intestinal parasite of humans and animals. Blastocystis comprises multiple subtypes (ST), based on variability within the small subunit ribosomal (SSU rRNA) RNA gene. Although full-length reference sequences of the SSU rRNA gene are a current requirement to name a novel Blastocystis subtype, full-length reference sequences are not currently available for all subtypes. In the present study, Oxford Nanopore Minion long-read sequencing was employed to generate full-length SSU rRNA sequences for seven new Blastocystis subtypes for which no full-length references currently exist. Phylogenetic analyses and pairwise distance matrixes were used to compare full-length and partial sequences, of the two regions most commonly used for subtyping. Analyses included Blastocystis nucleotide sequences obtained for subtypes (ST21, ST21-ST28) in this study and existing subtypes for which full-length reference sequences were available (ST1-ST17, ST29). The relationships and sequence variance between new and existing subtypes observed in analyses of different portions of the SSU rRNA gene are discussed. The full-length SSU rRNA reference sequences generated in this study provide essential new data to study and understand the relationships between the genetic complexity of Blastocystis and its host specificity, pathogenicity, and epidemiology. This information should be useful to other scientists and public health agencies working on Blastocystis.

Technical Abstract: Blastocystis is a common food- and water-borne intestinal protist parasite of humans and many other animals. Blastocystis comprises multiple subtypes (ST), based on variability within the small subunit ribosomal (SSU rRNA) RNA gene. Although full-length reference sequences of the SSU rRNA gene are a current requirement to name a novel Blastocystis subtype, full-length reference sequences are not currently available for all subtypes. In the present study, Oxford Nanopore Minion long-read sequencing was employed to generate full-length SSU rRNA sequences for seven new Blastocystis subtypes for which no full-length references currently exist: ST21, ST23, ST24, ST25, ST26, ST27, and ST28. Phylogenetic analyses and pairwise distance matrixes were used to compare full-length and partial sequences, of the two regions most commonly used for subtyping. Analyses included Blastocystis nucleotide sequences obtained in this study (ST21, ST21-ST28) and existing subtypes for which full-length reference sequences were available (ST1-ST17, ST29). The relationships and sequence variance between new and existing subtypes observed in analyses of different portions of the SSU rRNA gene are discussed. The full-length SSU rRNA reference sequences generated in this study provide essential new data to study and understand the relationships between the genetic complexity of Blastocystis and its host specificity, pathogenicity, and epidemiology.