Genome skimming is a low-cost and robust strategy to assemble complete mitochondrial genomes from ethanol preserved specimens in biodiversity studies.

Bruna Trevisan,Denis Jacob Machado,Daniel J.G Lahr,Daniel M.C Alcantara,Fernando P.L Marques

doi:10.7717/peerj.7543

Bruna Trevisan, Denis Jacob Machado + Show 3 more

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https://doi.org/10.7717/peerj.7543

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Abstract

Global loss of biodiversity is an ongoing process that concerns both local and global authorities. Studies of biodiversity mainly involve traditional methods using morphological characters and molecular protocols. However, conventional methods are a time consuming and resource demanding task. The development of high-throughput sequencing (HTS) techniques has reshaped the way we explore biodiversity and opened a path to new questions and novel empirical approaches. With the emergence of HTS, sequencing the complete mitochondrial genome became more accessible, and the number of genome sequences published has increased exponentially during the last decades. Despite the current state of knowledge about the potential of mitogenomics in phylogenetics, this is still a relatively under-explored area for a multitude of taxonomic groups, especially for those without commercial relevance, non-models organisms and with preserved DNA. Here we take the first step to assemble and annotate the genomes from HTS data using a new protocol of genome skimming which will offer an opportunity to extend the field of mitogenomics to under-studied organisms. We extracted genomic DNA from specimens preserved in ethanol. We used Nextera XT DNA to prepare indexed paired-end libraries since it is a powerful tool for working with diverse samples, requiring a low amount of input DNA. We sequenced the samples in two different Illumina platform (MiSeq or NextSeq 550). We trimmed raw reads, filtered and had their quality tested accordingly. We performed the assembly using a baiting and iterative mapping strategy, and the annotated the putative mitochondrion through a semi-automatic procedure. We applied the contiguity index to access the completeness of each new mitogenome. Our results reveal the efficiency of the proposed method to recover the whole mitogenomes of preserved DNA from non-model organisms even if there are gene rearrangement in the specimens. Our findings suggest the potential of combining the adequate platform and library to the genome skimming as an innovative approach, which opens a new range of possibilities of its use to obtain molecular data from organisms with different levels of preservation.

Highlights

Global loss of biodiversity is an ongoing process that concerns both local and global authorities (Vellend, 2017)
The DNA concentration obtained for P. longicrus was closer to the amount necessary to prepare the library, and its DNA was not diluted
The sequence on Illumina MiSeq resulted in a total of 13.98 mi PE reads for P. parvula and 16.82 mi PE reads for P. longicrus

Summary

Introduction

Global loss of biodiversity is an ongoing process that concerns both local and global authorities (Vellend, 2017). Challenges including habitat loss, overexploitation, climate change, and invasive species are far from a solution (Corlett, 2017; Vellend, 2017). Studies of biodiversity mainly involve traditional methods using morphological characters and molecular protocols predominantly by PCR-based methods. The PCR-based methods are often not successful in recovering genetic data of preserved organisms, due to the fragmented nature of old and poorly-preserved DNA (Heintzman et al, 2014; Timmermans et al, 2016). The lack of genomic resources such as well-established and optimized molecular markers and primers to delimit target amplicons for closely related species may hamper PCR-based methods for non-model organisms (Ekblom & Galindo, 2011; Tilak et al, 2015; Matos-Maraví et al, 2019)

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