Abstract
BackgroundIn the present study, we used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to obtain complete mitochondrial (mt) genomes of individual ticks and unprecedently performed precise annotation of these mt genomes. We aimed to: (1) develop a simple, cost-effective and accurate method for the study of extremely high AT-content mt genomes within an individual animal (e.g. Dermacentor silvarum) containing miniscule DNA; (2) provide a high-quality reference genome for D. silvarum with precise annotation and also for future studies of other tick mt genomes; and (3) detect and analyze mt DNA variation within an individual tick.ResultsThese annotations were confirmed by the PacBio full-length transcriptome data to cover both entire strands of the mitochondrial genomes without any gaps or overlaps. Moreover, two new and important findings were reported for the first time, contributing fundamental knowledge to mt biology. The first was the discovery of a transposon-like element that may eventually reveal much about mechanisms of gene rearrangements in mt genomes. Another finding was that Copy Number Variation (CNV) of Short Tandem Repeats (STRs) account for mitochondrial sequence diversity (heterogeneity) within an individual tick, insect, mouse or human, whereas SNPs were not detected. The CNV of STRs in the protein-coding genes resulted in frameshift mutations in the proteins, which can cause deleterious effects. Mitochondria containing these deleterious STR mutations accumulate in cells and can produce deleterious proteins.ConclusionsWe proposed that the accumulation of CNV of STRs in mitochondria may cause aging or diseases. Future tests of the CNV of STRs hypothesis help to ultimately reveal the genetic basis of mitochondrial DNA variation and its consequences (e.g., aging and diseases) in animals. Our study will lead to the reconsideration of the importance of STRs and a unified study of CNV of STRs with longer and shorter repeat units (particularly polynucleotides) in both nuclear and mt genomes.
Highlights
In the present study, we used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to obtain complete mitochondrial genomes of individual ticks and unprecedently performed precise annotation of these mt genomes
The present study focused on the genus Dermacentor belonging to Metastriata using ticks from four species (D. silvarum, D. nuttalli, D. marginatus and D. niveus)
Using specific primers (Table 1), each entire mt genome was amplified in two large segments: large segment 1 (L1) and large segment 2 (L2) or large segment 3 (L3) and large segment 4 (L4)
Summary
We used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to obtain complete mitochondrial (mt) genomes of individual ticks and unprecedently performed precise annotation of these mt genomes. Novel findings included the 3′ polyadenylation and possible 5′ m7G caps of rRNAs [1], the polycistronic transcripts [1], the antisense transcripts of all mt genes [1], and novel long non-coding RNAs (lncRNAs) [3] Based on these findings, we proposed the uninterrupted transcription of mammal mt genomes [3]. The second contribution concerned the use 5′ and 3′ end small RNAs (5′ and 3′ sRNAs) [4] to annotate mt genes to a resolution of 1 bp, subsequently dubbed “precise annotation” [5] Precise annotation of these accurate genomes led us to discover a novel 31-nt ncRNA in mammalian mt DNA [4] and that the copy numbers of tandem repeats exhibit great diversity within an E. fullo individual [5]. Precise annotation of human, chimpanzee, rhesus macaque and mouse mt genomes has been performed to study five Conserved Sequence Blocks (CSBs) in the mt D-loop region [6]; this led to a deep understanding of the mechanisms involved in the RNA-DNA transition and even the functions of the D-loop
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