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

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

doi:10.7717/peerj.7543

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

PeerJ. 2019 Sep 13:7:e7543. doi: 10.7717/peerj.7543. eCollection 2019.

Authors

Bruna Trevisan^#¹, Daniel M C Alcantara^#¹, Denis Jacob Machado^{1

2}, Fernando P L Marques¹, Daniel J G Lahr¹

Affiliations

¹ Department of Zoology, Institute of Biosciences, University of São Paulo, São Paulo, São Paulo, Brazil.
² Department of Bioinformatics and Genomics / College of Computing and Informatics, University of North Carolina at Charlotte, Charlotte, NC, United States of America.

^# Contributed equally.

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.

Keywords: Automated annotation; Automated assembly; Cestoda; Diptera; High-coverage genome; Mitochondrial genome; Poorly preserved samples.

Grants and funding

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP grants #2013/04585-3, #2016/20792-7, #2017/11063-4 and #2018/03534-0. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.