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# Mitochondrion
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Check available resources on mitochondrion and assembly


# Available mitochondrion genome complete

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| NCBI accession | project | sample | submission | reference | size (bp) |
|----------------|---------|--------|------|-----------|--------|
| [NC_042226](https://www.ncbi.nlm.nih.gov/nuccore/NC_042226.1) | [PRJNA541607](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA541607) | N/A  | 7-May-2019 | [Chen et al](https://www.tandfonline.com/doi/full/10.1080/23802359.2018.1507649) | 16,061 | 
| [MH536744](https://www.ncbi.nlm.nih.gov/nuccore/MH536744) | [PRJNA541607](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA541607) | N/A  | 25-Jun-2018 | [Chen et al](https://www.tandfonline.com/doi/full/10.1080/23802359.2018.1507649) | 16,061 |
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| [LR991693](https://www.ncbi.nlm.nih.gov/nuccore/LR991693.1) | [PRJEB42171](https://www.ncbi.nlm.nih.gov/bioproject/PRJEB42171) | [SAMEA7521635](https://www.ncbi.nlm.nih.gov/biosample/SAMEA7521635) | 25-Jan-2021 | N/A (Cambridge) | 22,664 |
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| [MT483700](https://www.ncbi.nlm.nih.gov/nuccore/MT483700.1) | N/A | N/A | 18-May-2020| N/A (Copenhagen) | 17,279 |
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* **NC_042226** and **MH536744** are identicals.
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* **LR991693** is extracted from the genome assembly **aRanTem1.1**
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* **MT483700** is partial.
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# Available nuclear genome complete

We can extract mitochondrion genome sequence from nuclear genome sequence.

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| Genome assembly | project | sample | submission | reference |
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|----------------|---------|--------|------|-----------|
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| [aRanTem1.1](https://www.ncbi.nlm.nih.gov/assembly/GCA_905171775.1) | [PRJEB42171](https://www.ncbi.nlm.nih.gov/bioproject/PRJEB42171) | [SAMEA7521635](https://www.ncbi.nlm.nih.gov/biosample/SAMEA7521635) | 28-Jan-2021 | N/A (Cambridge) |
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| [UCB_Rtem_1.0](https://www.ncbi.nlm.nih.gov/assembly/GCA_009802015.1/) | [PRJNA550264](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA550264/) | [SAMN12123141](https://www.ncbi.nlm.nih.gov/biosample/SAMN12123141/) | 27-Dec-2019 | N/A (Berkeley) |
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* The mitochondrion assembly **LR991693** is extracted from **aRanTem1.1**
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* No mitochondrion extracted from **UCB_Rtem_1** 
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* :exclamation: **UCB_Rtem_1** illumina raw reads necessary to extract mitogenome are not available https://www.ncbi.nlm.nih.gov/Traces/wgs/VIAC01
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# Designing mitochondrial baits 

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We considered the two high-quality mitochondrial genomes assemblies available in NCBI, MH536744 and MT483700. Despite being less properly assebmled, a third  non-annotated mitocondrion sequence (LR991693) was included here since it aligned correctly except for two regions of 3500 and 1500  bp. 
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We targeted the complete mitochondrion genome, including less conservative regions in both intragenic and coding genes. To that end, we run MAFFT to align the three above-mentioned mitochondial genomes using Geneious. Positions containing any gaps were masked and removed.
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Baits were designed as **180 base pairs length**, spread across the entire mitochondrial genome (no overlapping sequences as no need for a mitochondrial assembly) using [BaitDesigner](https://gatk.broadinstitute.org/hc/en-us/articles/360037069172-BaitDesigner-Picard-). The T7 promoter was added to the 5' end of the baits for later transcription using the [NEB kit E2040S](https://international.neb.com/products/e2040-hiscribe-t7-high-yield-rna-synthesis-kit#Product%20Information). Altogether, there was only one ambiguity character which was simply depreciated.

![Transcription of DNA baits to RNA](./Transcription.png)

As the mitochondiral genome of ***Rana temporaria*** is about 16,000 bp,  88 baits were designed per mitochondrion, we obtained a total of **286 baits**.
We synthetized the baits using a new technology for de-novo DNA synthesis available in [TWIST](https://www.twistbioscience.com/). 
We received a equimolar pool of the baits in one unique tube.
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# Hybridization baits and sequencing

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The baits will be received as DNA but will be rapidly transformed into RNA (to facilitate hybridization) following the same step described in the hyRAD protocol.
Hybridization will allow **5 to 10% divergence** between the DNA sequence and the bait sequence, allowing a maximum of 9 SNPs per sequence captured. 
The specificity of the baits will be adjusted according to the hybridization temperature, which means that two hybridization-captures are usually performed, one at 55°C and the other at 65°C (for more specificity). No species phylogenetically related to Rana temporaria are present on the ponds we sampled.  
Removal of non-Rana temporaria*** sequences will be done by post-computational processing and Miseq sequencing of our eDNA samples to screen for other species present in the eDNA sample. 
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