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

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.

Culicidae evolutionary history focusing on the Culicinae subfamily based on mitochondrial phylogenomics

“…gambiae was sequenced using the Sanger method on PCR amplified fragments 27 and further mitochondrial genomes were slowly sequenced along with whole genome projects. Currently, most studies have been using whole genome sequencing or PCR amplification followed by high throughput sequencing to characterize several mitogenomes at once 22 , 23 , 26 in a wide range of insect species showing promising results to reconstruct mitogenomes 32 , 43 . Here, we performed low-coverage whole genome sequencing to assemble and characterize the mitogenomes from seventeen mosquito species.…”

“…We were able to generate 16 new draft mitogenomes from Culicidae species belonging to eight different genera. This approach has already been used in other studies demonstrating a cost-effective way to recover mitogenomes for evolutionary studies 32 , 43 – 46 . Richter et al (2015) 32 suggested that a minimum of 10 million reads are needed to recover mitogenomes with higher coverage breadth and datasets having around 1 million reads usually generate highly incomplete mitogenomes.…”

Characteristics of Organellar Genomes and Nuclear Internal Transcribed Spacers in the Tertiary Relict Genus Dipelta and Their Phylogenomic Implications

“…The 2G raw data generated abundant DNA sequences of three genomes with high sequencing depth. These high-copy fractions of genomic DNA are beneficial to deal with the complex phylogenetic issues unresolved by the traditional DNA sequence fragments (Straub et al, 2012;Trevisan et al, 2019).…”

CONSULT: Accurate contamination removal using locality-sensitive hashing

“…Despite the decreased cost of whole-genome sequencing, carrying out large-scale cohort studies of non-human species using assembled genomes is still daunting (1). Low-cost sequencing projects remain an attractive alternative in biodiversity and ecological research (2, 3). Such studies can include a large number of samples sequenced at 1-2× read coverage, often called genome skims (4, 5, 6).…”