README.md

# Mitochondrion

Check available resources on mitochondrion and assembly


# Available mitochondrion genome complete

| 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 |
| [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 |
| [MT483700](https://www.ncbi.nlm.nih.gov/nuccore/MT483700.1) | N/A | N/A | 18-May-2020| N/A (Copenhagen) | 17,279 |

* **NC_042226** and **MH536744** are identicals.
* **LR991693** is extracted from the genome assembly **aRanTem1.1**
* **MT483700** is partial.


# Available nuclear genome complete

We can extract mitochondrion genome sequence from nuclear genome sequence.


| Genome assembly | project | sample | submission | reference |
|----------------|---------|--------|------|-----------|
| [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) |
| [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) |

* The mitochondrion assembly **LR991693** is extracted from **aRanTem1.1**
* No mitochondrion extracted from **UCB_Rtem_1** 
* :exclamation: **UCB_Rtem_1** illumina raw reads necessary to extract mitogenome are not available https://www.ncbi.nlm.nih.gov/Traces/wgs/VIAC01

# Designing mitochondrial baits 

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. 
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.
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.


# Hybridization baits and sequencing

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.