- Background: In 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. - Results: These annotations were confirmed by the PacBio full-length transcriptome data to cover both entire strands of the mitochondrial genomes without any gaps or overlaps. - The CNV of STRs in the protein-coding genes resulted in frameshift mutations in the proteins, which can cause deleterious effects. - 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.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - constructed the first quantitative transcription map of animal mt ge- nomes by sequencing the full-length transcriptome of the insect Erthesina fullo Thunberg [1] on the PacBio platform [2]. - this ultim- ately led to a deep understanding of the mechanisms in- volved in the RNA-DNA transition and even the functions of the D-loop.. - In the present study, we used long-PCR amplification coupled with Next-Generation Sequencing (NGS) to ob- tain complete mt genomes of individual ticks and per- formed precise annotation of these mt genomes. - Given that conventional mtDNA isolation and purification are not required in our method and in the Whole-Genome Sequencing (WGS) method, both the WGS method and our method are simple and cost-effective. - However, compared to the WGS method, our method has three main advantages: (1) errors in the assembly of mt ge- nomes caused by highly similar exogenous or nuclear se- quences [i.e., Nuclear Mitochondrial DNA (NUMT)] are avoided. - In the present study, we aimed to achieve the following research goals: (1) develop a simple, cost-effective and accurate method for the study of extremely high AT-content mt genomes within an in- dividual animal (e.g. - Using long-PCR and NGS to obtain complete mt genomes of individual ticks. - The type III mt genomes of individual ticks (Fig. - All the reference genomes of tick mitochondria read in the 5. - Using ~ 4 Gbp 2 × 150 DNA-seq data for each genome, the complete mt genomes of D. - Furthermore, R1 and R2 on L1 were validated using PCR amplification coupled with Sanger sequen- cing, separately, as the repeat units of R1 are the reverse complements of the repeat units of R2 (Fig. - Compari- son of the D. - silvarum mt genomes obtained by sequen- cing L3 and L4 with those obtained by sequencing L1 and L2 improved the accuracy of the DNA sequence.. - Gbp/1.5 Kbp) of the D. - silvarum mt genome (GenBank: MN347015) which was used as a ref- erence for precise annotation in the following studies.. - 1a): (1) the mt genomes of D.. - Translocated genes are reported in the same colour. - The type III mt genomes of ticks read clockwise in the 5. - These primers were designed to amplify large segments (L1, L2, L3 and L4) and short segments (CR1, CR2, R1 and R2) in the mt genomes of the genus Dermacentor . - Their PCR reaction conditions can be seen in the Methods. - Based on the results using 100 individual ticks from four species, the primers for L3 and L4 were optimized to amplify more species of the genus Dermacentor than those of L1 and L2. - silvarum mt genome (GenBank: MN347015). - each other, allowing for very rare Single Nucleotide Polymorphisms (SNPs) in the repeat units. - The mini- mum length of the repeat units of STRs is obviously 1 bp. - Polynucleotides and tandem repeats R1 and R2 had the same pattern of vari- ation in the D. - silvarum mt genome (below). - Precise annotation of the Dermacentor silvarum mt genome. - genome NC_026552.1 in the NCBI RefSeq database. - We performed precise annotation of the complete D. - silvarum mt genome (Table 2) using sRNA-seq data and confirmed these annotations using the PacBio full-length transcrip- tome data (Methods). - Although most of the new annota- tions were consistent with those of NC_026552.1, we corrected many errors in NC_026552.1, particularly in tRNAs, rRNAs, CR1, CR2, R1 and R2. - However, the Transcription Initiation Termination Sites (TISs) and the Transcription Termination Sites (TTSs) of the mt pri- mary transcripts of ticks are still not determined due to in- sufficient data available.. - CR1 and CR2 were determined in the D. - Another new finding was that the intergenic regions between tick mt tRNA genes are longer than those in mammals except a novel 31-nt ncRNA [4], which was generated in the gene order rearrangement of mammalian mt tRNA genes. - 3) were predicted to be two non- coding and non-transcriptional regions in the previous study [7]. - In the present study, however, they were proven to be transcribed on two strands. - individual, which confirmed a finding from our previous study of the E. - (4) in general, R1 sequences from individual ticks of one species comprised repeat units of one type and R1 sequences from individual ticks of the same spe- cies from different places could have different copy numbers. - and (5) of the four species of ticks we studied, D. - margina- tus, most of the R1s were composed of the type 3 units;. - however, a few had R1s composed of the types 1 and 2 hybrid units, noted as [R 34 ] l -[R 28 ] m -[R 34 ] n , where l, m and n represent the copy numbers. - 3 The transposon-like element in the Dermacentor silvarum mt genome. - All the mt genomes read in the 5. - R1 and R2 were determined to have 5 repeat units in the D. - Table 2 Precise annotations of the Dermacentor silvarum mt genome. - resulting in the insertion of [R 28 ] m into [R 34 ] l + n . - This confirmed our proposal of DNA-recombination events in a previous study of the E. - “tick box”—a degenerate 17-bp sequence motif that may be involved in the 3′ formation of ND1 and tRNA Glu transcripts in all major tick lineages . - A large translocated segment (LT1) spanning from ND1 to tRNA Gln was first reported in and the pres- ence of the “tick box” motif at both ends of this LT1 in- dicated its involvement in recombination events that are responsible for known Metastriata ticks [12–14]. - In the present study, LT1 was corrected to span R2, ND1, tRNA Leu , 16S rRNA, tRNA Val , 12S rRNA, CR1, tRNA Ile , tRNA Gln and R1 (Fig. - 3a) in the reference genome using precise annotations. - Given that nearly half of the human genome is various types of transposable elements that contain repetitive DNA sequences [19], we hypothesized that LT1 is a transposon, with R1 and R2 as invert re- peats (IRs) and genes from ND1 to tRNA Gln as insert se- quences (ISs). - To test our hypothesis, we sought structural variation (Methods) in the D. - Since LT1 inversions were rare, 4.1 Gbp DNA-seq data were generated to cover Gbp/9.58 Kbp) of L1 in the D. - As the dominant copy number was five for both R1 and R2, we used 34 × 5 STR to represent R1 and R2 in the D. - which is longer than the reads in the 2 × 150 bp DNA- seq data. - However, we did not obtain full-length sequences of LT1 due to sequence- length limitations in the DNA-seq data. - Copy number variation of STRs in the mt genomes within an individual animal. - We defined the STR position as the genomic position of the first nucleotide of the reference STR. - Importantly, it was found that almost all of the STRs had multiple variants, particularly those with copy num- bers greater than 5. - The detection of CNV of STRs was reliable, based on the following reasons: (1) PCR amplifi- cation and deep DNA sequencing produces a high signal-to-noise ratio in the detection of DNA variation;. - represent the major and minor coding strands of the mt genome, respectively. - Using a very strict parameter (Methods), we selected 20 STR positions in the D. - Almost all of the STRs were composed of A or T, except [G] 8 at pos- ition 1810. - All of the reference STRs and their variants had copy numbers greater than 5. - Among all of the vari- ants, 1 × n STR occurred much more frequently than m × n STR (m >. - Thirteen STR positions in the protein- coding genes had identical sequences between individuals, exhibiting a high degree of evolutionary conservation;. - however, the other seven STR positions in the tRNA and rRNA genes exhibited variation. - The CNV of STRs in the protein- coding genes resulted in frameshift mutations in the pro- teins, which may be deleterious [22]. - This finding inspired us to investigate if ani- mal cells have mechanisms to remove mitochondria con- taining deleterious mutations or inhibit the expression of the deleterious variants, as they can cause loss of function or diseases. - We had to compare the STR variants in the E. - In the allele column, insertions and deletions were noted as. - The numbers in the depth column are one-to-one corresponding to the variants in the allele column. - In particular, the use of SNPs is becoming dominant in the studies of mitochon- dria, e.g., mt heterogeneity. - However, most research focuses on SNPs, rather than CNV of STRs in mt genomes. - In the present study, we only detected CNV of STRs in mt ge- nomes within an individual animal and found that the alternative allele ratio was distributed from less than 0.01% to ~ 33%. - silvarum mt genome). - STRs located in regulatory, intron and transposon regions are beyond the scope of the present study. - Huntington’s disease, as one of the famous TRDs, occurs in the context of expanded glutamine [CAG] n repeats. - The telomeres at the ends of the chromosomes consist of [TTAGGG] n in vertebrates. - In the present study, we used long-PCR amplification coupled with NGS to obtain complete mt genomes of in- dividual ticks and performed precise annotation of these genomes. - The second finding may pave the way to an eventual understanding of the mechanisms of mt DNA variation. - This finding will encourage reconsideration of the importance of STRs as well as a unified study of CNV of STRs with shorter and longer repeat units in both nuclear and mt genomes.. - niveus) of the genus Derma- centor were collected from different places in China and were identified using a stereoscopic microscope accord- ing to [28]. - However, pre- cise annotations of the D. - To confirm the precise annotation of the D. - Statistics computing and graphics were conducted using the software R v2.15.3 the Bioconductor packages [32].. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - Using high-resolution annotation of insect mitochondrial DNA to decipher tandem repeats in the control region. - The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region. - Phylogenetic analysis of the mitochondrial genomes and nuclear rRNA genes of ticks reveals a deep phylogenetic structure within the genus Haemaphysalis and further elucidates the polyphyly of the genus Amblyomma with respect to Amblyomma sphenodonti and am. - Evolution of duplicate control regions in the mitochondrial genomes of metazoa: a case study with Australasian Ixodes ticks
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