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.

Keywords: Crayfish; Lobster; Long-read sequencing; Nanopore.

Fig. 1

The Caribbean spiny lobster Panulirus argus (left) and circular genome map of Panulirus argus mitochondrial DNA (right). The map is annotated and depicts 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rrnS [12S ribosomal RNA] and rrnL [16S ribosomal RNA]), 22 transfer RNA (tRNA) genes, and the putative control region. The inner circle depicts GC content along the genome

Fig. 2

Sequence errors per de-novo (Fyer and Unicycler) and reference-based assemblers (Rebaler) with and without ‘extra polishing’ with the software Medaka for the Caribbean spiny lobster Panulirus argus mitochondrial genome. All long-read assemblies were benchmarked against the Illumina short-read assembly with a coverage of 720x that served as a ‘gold’ standard in this study

Fig. 3

Annotation of de novo (Fyer and Unicycler) and reference-based (Rebaler) assemblies with and without ‘extra polishing’ with the software Medaka for the Caribbean spiny lobster Panulirus argus mitochondrial genome

Fig. 4

Mitophylogenomic analysis of the order Achelata, including mitochondrial genomes of Caribbean spiny lobster Panulirus argus assembled with long reads alone and short reads (‘gold standard’)

Fig. 5

Barcoding analysis of the order Achelata using a 500 bp fragment of the cox1 gene, including cox1 gene fragments retrieved from mitochondrial genomes of the Caribbean spiny lobster Panulirus argus assembled with long reads alone and short reads (‘gold standard’) plus 1884 other terminals belonging to the order Achelata retrieved from Genbank

Fig. 6

Bioinformatics pipeline to assemble the mitochondrial genome of the Caribbean spiny lobster using nanopore long reads