Abstract
Mitogenome sequences are highly desired because they are used in several biological disciplines. Their elucidation has been facilitated through the development of massive parallel sequencing, accelerating their deposition in public databases. However, sequencing, assembly and annotation methods might induce variability in their quality, raising concerns about the accuracy of the sequences that have been deposited in public databases. In this work we show that different sequencing methods (number of species pooled in a library, insert size and platform) and assembly and annotation methods generated variable completeness and similarity of the resulting mitogenome sequences, using three species of predaceous ladybird beetles as models. The identity of the sequences varied considerably depending on the method used and ranged from 38.19 to 90.1% for Cycloneda sanguinea, 72.85 to 91.06% for Harmonia axyridis and 41.15 to 93.60% for Hippodamia convergens. Dissimilarities were frequently found in the non-coding A+T rich region, but were also common in coding regions, and were not associated with low coverage. Mitogenome completeness and sequence identity were affected by the sequencing and assembly/annotation methods, and high within-species variation was also found for other mitogenome depositions in GenBank. This indicates a need for methods to confirm sequence accuracy, and guidelines for verifying mitogenomes should be discussed and developed by the scientific community.
Highlights
Mitochondrial DNA is used as a fundamental genetic marker in many research areas, including population genetics, evolutionary biology, phylogenetics and phylogeography, biodiversity studies, and molecular ecology [1,2,3]
Elucidation of mitogenomes relied on long range PCR (LR-PCR) plus primer walking coupled with Sanger dideoxy sequencing
There was variation in the number of reads mapped to the assembled mitogenomes across pipelines (Table 2), which was directly related to the coverage (Fig 2), but not related to the number of raw reads
Summary
Mitochondrial DNA (mtDNA, mitochondrial genome or mitogenome) is used as a fundamental genetic marker in many research areas, including population genetics, evolutionary biology, phylogenetics and phylogeography, biodiversity studies, and molecular ecology [1,2,3]. Insect mitochondrial genomes are mostly about 15–22 kbp, circular, double-stranded DNAs that encode a set of 37 genes and a large control region (designated as the A+T-rich region in insects) [3,4]. The A+T-rich region is responsible for regulating. Mitogenome sequence accuracy using different elucidation methods transcription and replication [5] and accounts for much of the mitogenome size variation across insect species [6]. Elucidation of mitogenomes relied on long range PCR (LR-PCR) plus primer walking coupled with Sanger dideoxy sequencing. New approaches, using next-generation sequencing (NGS) have become common, including LR-PCR plus NGS, RNA sequencing (RNAseq) plus gap filling, and direct shotgun sequencing [3,7,8,9]
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