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

Juan Antonio Baeza

doi:10.14293/s2199-1006.1.sor-.pp7raay.v1

Abstract

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.

Highlights

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
I tested if a mitochondrial genome can be sequenced and assembled from long read nanopore sequencing data exclusively using both a de novo and a reference-based strategy

Summary

Introduction

I tested if a mitochondrial genome can be sequenced and assembled from long read nanopore sequencing data exclusively using both a de novo and a reference-based strategy. I explored the quality (i.e., accuracy) of the long read-based assembled genomes by comparing them to a 'gold' standard mitochondrial genome retrieved from the same individual but generated using short read Illumina sequencing data. Sequence accuracy was explored for different long read assembly pipelines with multiple metrics including completeness, identity, and coverage. I tested if the de novo and reference-based long read assemblies are useful for mitophylogenomic and barcoding research. I assessed if long read assemblies do have phylogenomic information that permit to reliably identify the sequenced specimen and distinguish it from other closely and distantly related species in the same genus, family, and superorder

Objectives

Methods

Conclusion