Dataset Information

Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus.

ABSTRACT: Background: Whole mitogenomes or short fragments (i.e., 300-700?bp of the cox1 gene) are the markers of choice for revealing within- and among-species genealogies. Protocols for sequencing and assembling mitogenomes include 'primer walking' or 'long PCR' followed by Sanger sequencing or Illumina short-read low-coverage whole genome (LC-WGS) sequencing with or without prior enrichment of mitochondrial DNA. The aforementioned strategies assemble complete and accurate mitochondrial genomes but are time consuming and/or expensive. In this study, I first tested whether mitogenomes can be sequenced from long-read nanopore sequencing data exclusively. Second, I explored the accuracy of the long-read assembled genomes by comparing them to a 'gold' standard reference mitogenome retrieved from the same individual using Illumina sequencing. Third and lastly, I tested if the long-read assemblies are useful for mitophylogenomics and barcoding research. To accomplish these goals, I used the Caribbean spiny lobster Panulirus argus, an ecologically relevant species in shallow water coral reefs and target of the most lucrative fishery in the greater Caribbean region.

Results: LC-WGS using a MinION ONT device and various de-novo and reference-based assembly pipelines retrieved a complete and highly accurate mitogenome for the Caribbean spiny lobster Panulirus argus. Discordance between each of the long-read assemblies and the reference mitogenome was mostly due to indels at the flanks of homopolymer regions. Although not 'perfect', phylogenetic analyses using entire mitogenomes or a fragment of the cox1 gene demonstrated that mitogenomes assembled using long reads reliably identify the sequenced specimen as belonging to P. argus and distinguish it from other related species in the same genus, family, and superorder.

Conclusions: This study serves as a proof-of-concept for the future implementation of in-situ surveillance protocols using the MinION to detect mislabeling in P. argus across its supply chain. Mislabeling detection will improve fishery management in this overexploited lobster. This study will additionally aid in decreasing costs for exploring meta-population connectivity in the Caribbean spiny lobster and will aid with the transfer of genomics technology to low-income countries.

SUBMITTER: Baeza JA

PROVIDER: S-EPMC7726883 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus.

Baeza J Antonio JA

BMC genomics 20201209 1

<h4>Background</h4>Whole mitogenomes or short fragments (i.e., 300-700 bp of the cox1 gene) are the markers of choice for revealing within- and among-species genealogies. Protocols for sequencing and assembling mitogenomes include 'primer walking' or 'long PCR' followed by Sanger sequencing or Illumina short-read low-coverage whole genome (LC-WGS) sequencing with or without prior enrichment of mitochondrial DNA. The aforementioned strategies assemble complete and accurate mitochondrial genomes b ...[more]

PMID: 33297960

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Project description:BACKGROUND:Crustaceans express several classes of receptor genes in their antennules, which house olfactory sensory neurons (OSNs) and non-olfactory chemosensory neurons. Transcriptomics studies reveal that candidate chemoreceptor proteins include variant Ionotropic Receptors (IRs) including both co-receptor IRs and tuning IRs, Transient Receptor Potential (TRP) channels, Gustatory Receptors, epithelial sodium channels, and class A G-protein coupled receptors (GPCRs). The Caribbean spiny lobster, Panulirus argus, expresses in its antennules nearly 600 IRs, 17 TRP channels, 1 Gustatory Receptor, 7 epithelial sodium channels, 81 GPCRs, 6?G proteins, and dozens of enzymes in signaling pathways. However, the specific combinatorial expression patterns of these proteins in single sensory neurons are not known for any crustacean, limiting our understanding of how their chemosensory systems encode chemical quality. RESULTS:The goal of this study was to use transcriptomics to describe expression patterns of chemoreceptor genes in OSNs of P. argus. We generated and analyzed transcriptomes from 7 single OSNs, some of which were shown to respond to a food odor, as well as an additional 7 multicell transcriptomes from preparations containing few (2-4), several (ca. 15), or many (ca. 400) OSNs. We found that each OSN expressed the same 2 co-receptor IRs (IR25a, IR93a) but not the other 2 antennular coIRs (IR8a, IR76b), 9-53 tuning IRs but only one to a few in high abundance, the same 5 TRP channels plus up to 5 additional TRPs, 12-17 GPCRs including the same 5 expressed in every single cell transcriptome, the same 3?G proteins plus others, many enzymes in the signaling pathways, but no Gustatory Receptors or epithelial sodium channels. The greatest difference in receptor expression among the OSNs was the identity of the tuning IRs. CONCLUSIONS:Our results provide an initial view of the combinatorial expression patterns of receptor molecules in single OSNs in one species of decapod crustacean, including receptors directly involved in olfactory transduction and others likely involved in modulation. Our results also suggest differences in receptor expression in OSNs vs. other chemosensory neurons.

Project description:The spiny lobster, Panulirus argus, has two classes of chemosensilla representing "olfaction" and "distributed chemoreception," as is typical for decapod crustaceans. Olfactory sensilla are found exclusively on antennular lateral flagella and are innervated only by olfactory receptor neurons (ORNs) that project into olfactory lobes organized into glomeruli in the brain. Distributed chemoreceptor sensilla are found on all body surfaces including the antennular lateral flagella (LF) and walking leg dactyls (dactyls), and are innervated by both chemoreceptor neurons (CRNs) and mechanoreceptor neurons that project into somatotopically organized neuropils. Here, we examined expression of three classes of chemosensory genes in transcriptomes of the LF (with ORNs and CRNs), dactyls (with only CRNs), and brain of P. argus: Ionotropic Receptors (IRs), which are related to ionotropic glutamate receptors and found in all protostomes including crustaceans; Gustatory Receptors (GRs), which are ionotropic receptors that are abundantly expressed in insects but more restricted in crustaceans; and Transient Receptor Potential (TRP) channels, a diverse set of sensor-channels that include several chemosensors in diverse animals. We identified 108 IRs, one GR, and 18 homologues representing all seven subfamilies of TRP channels. The number of IRs expressed in the LF is far greater than in dactyls, possibly reflecting the contribution of receptor proteins associated with the ORNs beyond those associated with CRNs. We found co-receptor IRs (IR8a, IR25a, IR76b, IR93a) and conserved IRs (IR21a, IR40a) in addition to the numerous divergent IRs in the LF, dactyl, and brain. Immunocytochemistry showed that IR25a is expressed in ORNs, CRNs, and a specific type of cell located in the brain near the olfactory lobes. While the function of IRs, TRP channels, and the GR was not explored, our results suggest that P. argus has an abundance of diverse putative chemoreceptor proteins that it may use in chemoreception.

Dataset Information

Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus.

Publications

Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and barcoding research using the Caribbean spiny lobster Panulirus argus.

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