Yes, we can use it: a formal test on the accuracy of low-pass nanopore long-read sequencing for mitophylogenomics and ba
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METHODOLOGY ARTICLE
Open Access
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 J. Antonio Baeza1,2,3
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. (Continued on next page)
Correspondence: [email protected] 1 Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634, USA 2 Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce, Florida 34949, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted
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