Geli-Cruz, Orlando J.

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    Nanopore sequencing for the metagenomic analysis of the gut microbiota of the millipede Anadenobolus monilicornis
    (2019-05-07) Geli-Cruz, Orlando J.; Van-Dam, Alex; College of Arts and Sciences - Sciences; Santos-Flores, Carlos; Cafaro, Matías J.; Department of Biology; Macchiavelli, Raúl E.
    Millipedes act as important components of leaf litter decomposition in terrestrial ecosystems, thanks in part to the microbial diversity in their guts. However, millipedes and their gut microbiota are understudied, compared to other arthropods. For this reason, we designed a protocol for the extraction, sequencing, and shotgun metagenomic analysis of the gut of Anadenobolus monilicornis. We collected specimens of A. monilicornis from different municipalities of Puerto Rico. We extracted their guts and sequenced the DNA with Oxford Nanopore Technologies’ MinION nanopore sequencer, then analyzed the data using the programs Phylosift and MEGAN6, and the MG-RAST online server. From our two successful samples from the municipalities of Mayagüez and Rincón, we obtained a total of 87,110 and 99,749 reads, respectively. We found that many of our assigned bacteria reads were annotated to the phyla Proteobacteria, Bacteroidetes, and Firmicutes; the Mayagüez sample had much more Chlamydiae representation, however. Sampled eukaryote phyla include Arthropoda, Streptophyta, and Chordata. Judging by the fact that some of the reads were annotated as belonging to Chordata, coupled with the lack of Nematoda and Ascomycota reads, we concluded that some missannotation may have occurred. We would need a larger sample size to better identify the intestinal microbial taxa, as we were setback by the landfall of Hurricane Maria in 2017. Considering our small sample size, however, we were able to develop an initial fingerprint of the millipede intestinal microbiota using shotgun metagenomics and nanopore sequencing.