Detection of Nudibranch Chromodoris quadricolor associated microbiome using two DNA extraction methods

Abstract

Article history: Received 27 April 2021 Received in revised form 25 May 2021 Accepted 26 May 2021 Available online 1 June 2021 DNA extraction with high quality is critical to all molecular genetic analyses. However, obtaining DNA from microbes associated with animals is challenging. Despite the availability of various DNA extraction kits in the markets, no studies were conducted to date to evaluate their potential for the invertebrates such as nudibranch, one of Mollusca. This study compared the Quick—DNA Fungal/Bacterial Miniprep Kit and CTAB methods and tested them across four samples of Chromodoris quadricolor gut and skin tissues. The universal bacterial primers 331f and 797r and the animalspecific primers LCO1490-JJ and HCO2198-JJ were used to amplify the 16S rRNA gene and partial mitochondrial cytochrome oxidase I gene using extracted DNA as a template. The DNA and PCR products’ quality and concentration were verified with agarose gel and Nanodrop, respectively. The two methods quality assessed using the deep pyrosequencing of the 16S rRNA gene in capturing a more diverse microbiome. The highest yield and purity (over 1000 ng / μL) were obtained with the CTAB method, while it was not exceeded 260 ng/μL with Quick DNA kit and display high purity. Also, 16S rRNA community amplicon sequencing revealed that the CTAB way could catch more diverse bacterial groups. The most efficient method of DNA extraction was CTAB, as it achieved both high concentration and purity.