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

Whole mitogenomes or short fragments (e.g., 300-700 bp of the cox1 gene) are 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 low-coverage whole genome (LCWGS) sequencing with or without prior mitochondrial enrichment and Illumina sequencing. The aforementioned strategies assemble complete and accurate mitochondrial genomes but are time consuming and/or expensive. In this study, I tested first if 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 individualusing Illumina sequencing. LC-WGS using a MinION ONT device and various de-novo and reference-based assembly pipelines did retrieve 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 can reliably identify the sequenced specimen as belonging to P. argus and distinguish it from other closely and distantly related species in the same genus, family, and superorder. This study serves as a proof-of-concept for the future implementation of in-situ surveillance protocols using the MinION so 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 and developing countries.

Condición nutricional de la langosta, Panulirus argus (Decapoda: Palinuridae): 60 años de índices extensivos y variación espacio-temporal en Cubas

Introducción: En el hábitat natural, el estado nutricional insatisfactorio de un número elevado de ejemplares es expresión de la influencia de factores desfavorables. Objetivo: Determinar variaciones espacio-temporales en el estado nutricional de langostas Panulirus argus, relacionarlas con variaciones reportadas en la comunidad bentónica y demostrar, mediante la aplicación de indicadores analíticos y morfométricos, que la variación de factores ambientales afecta a ambos grupos de índices; y que no todos los índices de condición nutricional evidencian por igual el impacto generalizado del proceso de deterioro ambiental en este golfo. Métodos: Las langostas se capturaron en seis áreas de pesca. La condición nutricional se estimó mediante tres índices no destructivos, poco costosos y de fácil y rápida aplicación (extensivos): índice de refracción de la hemolinfa (IRH), relación peso total / largo total = Klt, y factor de condición (FCA). Las variaciones a largo plazo (60 años) se determinaron mediante índices morfométricos (Klt y FCA). Se analizaron datos de los períodos 1963-1964 (N = 29 001), 1983-1993 (N = 3 123) y 2011-2017 (N = 3 600), separándolos en épocas de Lluvia y Seca. Resultados: En todos los períodos la condición nutricional varió significativamente entre áreas, pero sin similitudes entre períodos. Esto indica que los factores que impactan en el estado nutricional tienen una influencia estocástica más característica de factores ambientales. Aunque los tres índices fueron menores en 2017, sólo IRH disminuyó gradualmente entre 2011 y 2017, lo que sugiere que este índice, y los morfométricos, expresan diferente información. Todas las correlaciones fueron estadísticamente significativas, los coeficientes más altos se establecieron entre los índices morfométricos. El peso total y el Klt no mostraron diferencias entre Seca y Lluvia. Sin embargo, IRH y FCA resultaron mayores en Seca, hecho que se constató para cada sexo. FCA y Klt no presentaron diferencias entre el período 1963-1964 y el período 1983-1993, pero aumentaron (P < 0.05) en el período 2011-2017, lo que, dado el carácter morfométrico de estos índices, se atribuye a la presencia de langostas de mayor peso en las áreas de pesca. Conclusiones: Los menores valores en 2017 y la tendencia gradual al decrecimiento de IRH, corroboran el carácter generalizado del deterioro ambiental en el golfo de Batabanó, lo cual no fue igualmente expresado por los tres índices.

Genome survey sequencing of the Caribbean spiny lobster Panulirus argus: Genome size, nuclear rRNA operon, repetitive elements, and microsatellite discovery

Background Panulirus argus is an ecologically relevant species in shallow water hard-bottom environments and coral reefs and target of the most lucrative fishery in the greater Caribbean region. Methods This study reports, for the first time, the genome size and nuclear repetitive elements, including the 45S ribosomal DNA operon, 5S unit, and microsatellites, of P. argus. Results Using a k-mer approach, the average haploid genome size estimated for P. argus was 2.17 Gbp. Repetitive elements comprised 69.02% of the nuclear genome. In turn, 30.98% of the genome represented low- or single-copy sequences. A considerable proportion of repetitive sequences could not be assigned to known repeat element families. Taking into account only annotated repetitive elements, the most frequent belonged to Class I-LINE which were noticeably more abundant than Class I-LTR-Ty- 3/Gypsy, Class I-LTR-Penelope, and Class I-LTR-Ty-3/Bel-Pao elements. Satellite DNA was also abundant. The ribosomal operon in P. argus comprises, in the following order, a 5′ ETS (length = 707 bp), ssrDNA (1,875 bp), ITS1 (736 bp), 5.8S rDNA (162 bp), ITS2 (1,314 bp), lsrDNA (5,387 bp), and 3′ ETS (287 bp). A total of 1,281 SSRs were identified.

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

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.