TABLE 2.
Performance of our modified 16S rRNA gene nanopore sequencing protocol and NanoCLUST-based classification on confirmed bacterium- and filarial worm-positive controlsa
| Pathogen(s) present | Sample type | NanoCLUST classification | NCBI accession no. | Length (bp) | Identity (%) | No. of reads |
|---|---|---|---|---|---|---|
| Bacterial | ||||||
| Anaplasma phagocytophilum | Slides coated with cultured organisms | Anaplasma phagocytophilum | CP006618.1 | 1,422 | 99.93 | 3,297 |
| Anaplasma platys | Canine blood | Anaplasma platys | CP046391.1 | 1,422 | 100 | 1,007 |
| Bartonella henselae | Flea hemolymph | Bartonella henselae | CP020742.1 | 1,446 | 100 | 35,889 |
| Borrelia burgdorferi | Cell culture | Borrelia burgdorferi | CP031412.1 | 1,430 | 100 | 46,843 |
| “Candidatus Mycoplasma haematoparvum” | Canine blood | “Candidatus Mycoplasma haematoparvum” | AY383241.1 | 1,417 | 99.79 | 1,967 |
| Ehrlichia canis | Canine blood | Ehrlichia canis | CP025749.1 | 1,420 | 100 | 5,103 |
| Ehrlichia chaffeensis | Slides coated with cultured organisms | Ehrlichia chaffeensis | NR_074500.2 | 1,420 | 100 | 2,129 |
| Leptospira biflexa | Cell culture | Leptospira biflexa serovar Patoc | CP000786.1 | 1,417 | 100 | 34,669 |
| Mycoplasma haemocanis | Canine blood | Mycoplasma haemocanis | CP003199.1 | 1,354 | 99.79 | 217 |
| Orientia tsutsugamushi | Cell culture | Orientia tsutsugamushi | LS398550.1 | 1,417 | 100 | 67 |
| Rickettsia australis | Cell culture | Rickettsia australis | CP003338.1 | 1,418 | 100 | 16,231 |
| Rickettsia conorii | Cell culture | Rickettsia conorii | NR_074480.2 | 1,421 | 100 | 6,010 |
| Rickettsia felis | Cell culture | Rickettsia felis | CP000053.1 | 1,413 | 100 | 23,665 |
| Rickettsia honei | Cell culture | Rickettsia honei | NR_025967.1 | 1,417 | 99.86 | 21,963 |
| Rickettsia typhi | Cell culture | Rickettsia typhi | LS992663.1 | 1,420 | 100 | 39,097 |
| Anaplasma platys and Mycoplasma haemocanis | Canine blood | Anaplasma platys and Mycoplasma haemocanis | CP046391.1, CP003199.1 | 1,422, 1,354 | 99.93, 99.79 | 193, 52 |
| Anaplasma platys, Mycoplasma haemocanis, and E. canis | Canine blood | Anaplasma platys, Mycoplasma haemocanis, and Ehrlichia canis | CP046391.1, CP003199.1, CP025749.1 | 1,422, 1,354, 1,420 | 100, 99.79, 100 | 1,620, 179, 43 |
| Filarial worm(s) | ||||||
| Brugia malayi* | Canine blood | Wolbachia endosymbiont of Dirofilaria repens, Brugia malayi/Brugia pahangi, and Pseudolynchia canariensis | AJ276500.1, CP050521.1/CP034333.1, DQ115537.1 | 1,394, 1,414, 1,410 | 99.6, 99.93, 99.36 | 15,634, 281, 55 |
| Dirofilaria sp. Hong Kong genotype* | Canine blood | Wolbachia endosymbiont of Brugia malayi/Brugia pahangi | CP050521.1/CP034333.1 | 1,416 | 99.93 | 1,912 |
| Dirofilaria immitis | Canine blood | Wolbachia endosymbiont of Dirofilaria immitis | CP046578.1 | 1,421 | 100 | 520 |
a
Classification information shows the top hit(s), identity, and length of the relevant bacterial pathogen consensus sequence, as generated by the NanoCLUST pipeline, when classified using BLASTn with the GenBank database. Asterisks indicate positive controls that had multiple microfilaria morphologies observed under the microscope, indicative of a coinfection, but the result identified via cPCR and Sanger sequencing is given. Note that results in columns 4 to 7 correspond, respectively, to the data given in column 3, i.e., the results of the NanoCLUST pipeline.