Commit 7c3446da authored by Carrasquer's avatar Carrasquer
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Update README.md

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......@@ -33,12 +33,15 @@ We can extract mitochondrion genome sequence from nuclear genome sequence.
# Designing mitochondrial baits
We will consider the two mitochondrial genomes available in NCBI (see behind).
We will target less conservative regions in both intragenic and coding genes.
Baits will be designed as **180 base pairs length**, spread across the entire mitochondrial genome (no overlapping sequences as no need for a mitochondrial assembly).
As the mitochondiral genome of ***Rana temporaria*** is about 16,000 bp, a total of about **90 baits will be designed**.
We will synthetize the baits using a new technology for de-novo DNA synthesis available in [TWIST](https://www.twistbioscience.com/).
We will be receive the baits in one unique tube.
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
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