Development of a PCR-free marine micro-eukaryotic biodiversity assessment methodology

Abstract

In order to avoid the PCR bias and increase the efficiency of sequencing efforts targeted to marker sequences, we extracted environmental DNA from not whole cells including nucleus but subcellular organelles in environmental samples because nucleus DNA which is not sufficient genetic marker sequences compared with those from subcellular organelles. To validate our method, we collected two environmental samplesfrom Y amido (Yellow sea) and Baealdo (South sea) in South Korea. Furthermore, to test the taxonomic capturing capacity of our mitochondrial metagenomics method, we compared them to the results of DNA metabarcoding method employing four primary barcodes (COi, COI-5P, tufA and rbcL). From next-generation sequencing (NGS) libraries for DNA metabarcoding and mitochondrial metagenomics methods, in total we generated about 23.7 Gbp sequences. As a result, we conclude that the number of species by our mitochondrial metagenomics method was about 6-fold (1,034 species [776 genera] versus 165 species [71 genera] in Y amido sample and 1,017 species [718 genera] versus 252 species [130 genera] in Baealdo sample) higher than those by DNA metabarcoding method. These results were independent to aberration from the difference of raw-data size between two methods. We believe this method will present a useful alternative approach for evaluating marine environments by biodiversity. nucleus DNA which is not sufficient genetic marker sequences compared with those from subcellular organelles. To validate our method, we collected two environmental samplesfrom Y amido (Yellow sea) and Baealdo (South sea) in South Korea. Furthermore, to test the taxonomic capturing capacity of our mitochondrial metagenomics method, we compared them to the results of DNA metabarcoding method employing four primary barcodes (COi, COI-5P, tufA and rbcL). From next-generation sequencing (NGS) libraries for DNA metabarcoding and mitochondrial metagenomics methods, in total we generated about 23.7 Gbp sequences. As a result, we conclude that the number of species by our mitochondrial metagenomics method was about 6-fold (1,034 species [776 genera] versus 165 species [71 genera] in Y amido sample and 1,017 species [718 genera] versus 252 species [130 genera] in Baealdo sample) higher than those by DNA metabarcoding method. These results were independent to aberration from the difference of raw-data size between two methods. We believe this method will present a useful alternative approach for evaluating marine environments by biodiversity.