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Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellet’s whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. In our comparison of extraction methods, the Salting Out protocol was the least expensive, produced the highest DNA yields, produced consistent high DNA quality, and had low toxicity. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellet’s whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina NovaSeq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies.
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Full 18S metabarcoding of environmental samples of various substrates with Nanopore sequencing. v2
We describe here a flexible protocol for eDNA metabarcoding with Oxford Nanopore's MinIon MK1C platform from sampling to sequencing. The first section summarizes some key steps of sampling and sample preservation for both aquatic and terrestrial environments. The second one describes the DNA extraction protocol with the DNeasy PowerSoil Pro Kit of Qiagen for different types of samples (i.e., soil, liquid and dead plant materials). Our DNA amplification and bead purification protocols are characterized in the third section. Finally, the library prep with the Native amplicon Barcoding Kit 96 V14 (SQK-NBD114.96) and sequencing with the R10.4.1 flow cells (FLO-MIN114) and MinION Mk1C device are presented in the last sections. This protocol has been optimized for protists (microbial eukaryotes) and 18S marker, but should be easily adjustable for other organisms, by modifying the sampling and DNA extraction sections, or other markers, even longer ones such as the full ribosomal operon, if need be. Our goal is to teach/train researchers from different fields and different expertise on obtaining nanopore sequences from environmental samples by guiding them from protocol to protocol, focusing on the key steps, and informing them of the expected results based on our 3 runs performed so far. A comprehensive bioinformatic pipeline to treat the data produced, as well as a methodological article discussing this method and the best ways to use it are in preparation.
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- Award ID(s):
- 2100888
- PAR ID:
- 10574811
- Publisher / Repository:
- Protocols.io
- Date Published:
- Format(s):
- Medium: X
- Institution:
- Protocols.io
- Sponsoring Org:
- National Science Foundation
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- https://doi.org/10.17504/protocols.io.3byl4qe48vo5/v2
