Demonstration of a basic mitochondrial enrichment method to produce the complete mitochondrial genome sequence of the en
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TECHNICAL NOTE
Demonstration of a basic mitochondrial enrichment method to produce the complete mitochondrial genome sequence of the endangered North Atlantic right whale (Eubalaena glacialis) J. S. Allwood1 · M. K. Scheible1 · S. A. Faith1
Received: 13 July 2017 / Accepted: 28 August 2017 © Springer Science+Business Media B.V. 2017
Abstract The North Atlantic right whale is one of only three species within the Eubalaena genus, a group heavily targeted by the whaling industry prior to the twentieth century. All three species (Eubalaena australis, Eubalaena japonica and Eubalaena glacialis) are CITES listed (Appendix I), with the latter two species listed as endangered (IUCN, v 2017-1). The mitochondrial genome of the first two species have been sequenced and are publicly available, while E. glacialis was unavailable. Here we present the complete mitochondrial genome sequence of E. glacialis, while also detailing a straight forward mitochondrial enrichment method that facilitates analysis using next generation sequencing. This method simultaneously allows for efficient sequencing of the mitochondrial genome, while demonstrating quality sequence coverage that is even across the genome. This enrichment illustrates a marked improvement in sequence depth compared to that achieved when performing whole genome sequencing using a standard DNA extraction.
* J. S. Allwood [email protected] M. K. Scheible [email protected] S. A. Faith [email protected] 1
Department of Molecular Biomedical Sciences and The Forensic Sciences Institute, North Carolina State University, 1060 William Moore Dr., Raleigh, NC 27607, USA
Keywords Mitochondrial enrichment · Eubalaena glacialis · Mitochondrial genome · Next generation sequencing · Bioinformatics Blood preserved in EDTA from a North Atlantic right whale (New England Aquarium Identification: Eg#3710) was provided by the Center for Marine Sciences and Technology (CMAST, NCSU, NC, USA; Case report CALO2009-01, Harms et al. 2014). The standard DNA extraction [extracted previously using QIAamp Mini Blood Kit (Qiagen, Hilden, Germany)] used the same stored starting material as the enriched extraction undertaken here. The mitochondrial enrichment method was adapted from Ahmad et al. (2007) and Coutinho et al. (2017) for smaller starting volumes of blood (milliliters to microliters), to allow for greater application in wildlife research studies. TKM1 buffer was added in a 1:1 ratio to blood (250 µl each), with 1% volume of IGEPAL, and incubated at room temperature for 10 min, followed by a 20 min centrifugation at 800×g. Supernatant was retained and this step was repeated using the same quantities and centrifugation conditions applied as above. The retained supernatant from both TKM1 steps were combined and centrifuged for 20 min at 15,000×g. The resulting pellet was briefly washed twice in TKM1 and suspended in 500 µl TKM2 with 100 µl of 10% SDS and incubated overnight at 55 °C. Proteins were salted out with the addition of 400 µl of 3M NaCl, followed by centrifugation for 20 min at 1
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