Computational protocol: Mitochondrial Genome Assemblies of Elysia timida and Elysia cornigera and the Response of Mitochondrion-Associated Metabolism during Starvation

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[…] Mitochondrial (mt) genomes were primarily assembled from RNA-Seq data () using Sequencher (Sequencher v. 5.3, Gene Codes Corporation, USA) and standard assembly settings. In addition, continuous stretches of ∼6.5 kb of E. timida and ∼12 kb of E. cornigera mitochondrial DNA were sequenced by primer walking to close gaps and compare RNA and DNA sequences. Genomic DNA was extracted with Plant DNAzol (ThermoFisher) and Phusion High-Fidelity DNA polymerase (New England Biolabs) used for standard PCR reactions. Amplification products were sequenced and fed into the Sequencher assembly. The sequences were found to be close to identical—but to 100% in terms of contiguity—to those of the sequenced RNA, with only occasional differences in base calls (about 3 per 1kbp), but no larger gaps indicating potential introns. Due to the nature of the samples it is not possible to distinguish between, for example, single-nucleotide polymorphisms or RNA editing, but if the latter is occurring at all, frequencies would be marginal. The average sequence coverage was 508,168 and 210,540 for E. timida and E. cornigera, respectively. Gene annotation was performed using MITOS () and Geneious 8.0.3 (Biomatters, New Zeeland, ) with the mt genomes of Elysia chlorotica (), Thuridilla gracilis (), and Placida sp. () as references. Mitochondrial genome maps were generated using Organellar GenomeDRAW (). [...] For every protein-coding gene of the mitochondria we performed individual amino acid sequence alignments with Geneious 8.0.3 (Biomatters, New Zeeland, ) and using Siphonaria pectinata (AY345049) as the outgroup. Alignments were performed using Fast Fourier Transform (MAFFT; ) with the G-INSI mode, inspected by Aliscore () and conspicuous sites removed. All individual alignments were concatenated and a phylogenetic reconstruction performed using RaxML () with the LG + I+G + F model (four discrete gamma categories and sites), as suggest by ProtTest analyses (), and 1,000 bootstrap replicates. […]

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