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Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota

Citation

Chariton, Anthony; Pansu, Johan; Hose, Grant; Chapman, Michelle (2021), Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota, Dryad, Dataset, https://doi.org/10.5061/dryad.kwh70rz34

Abstract

Monitoring sediment biota is an essential step for the quality assessment of aquatic ecosystems. Environmental DNA-based approaches for biomonitoring are increasing in popularity; yet, commercial kits and protocols for extracting total DNA from sediments remain expensive and time-consuming. Furthermore, they can accommodate only small amounts of sediments, potentially preventing an adequate representation of local biodiversity, especially for macro-organisms. Here, we assessed the reliability of a cost- and time- effective extracellular DNA extraction approach, able to account for large volumes of starting material, for characterising bacterial, eukaryote and metazoan communities in three sedimentary environments. DNA concentrations extracted with the extracellular approach were at least similar to those obtained with the commercial kit. Local diversity estimates were not biased towards any particular extraction method, although specific responses were observed depending of the sediment type. Community composition and beta-diversity patterns were moderately affected by the extraction approach and the initial amount of starting material; differences being more important for macro- than micro-organisms. Thus, the extracellular DNA approach appears as robust and efficient as those based on the commercially-available kit for biomonitoring sedimentary communities. Its relatively low cost and faster processing time make it a promising alternative for large-scale ecological assessments of aquatic environments.

Methods

This study compares the composition of benthic communites (16S, 18S and CO1) from freshwater, estuarine and marine sediments extracted using two different approaches: commercial kits (intra and extra cellular DNA) and a phosphate buffer (extracelluar DNA). Comparisons are made using 1 and 10 grams of starting material, with the phosphate buffer also performed using 200g of sediment. 

 

Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. 
Data were filtered according to the filtering procedure described in Pansu et al. (2021): ‘Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota’ (Marine & Freshwater Research). 
Taxonomic assignment was made against a set of curated 16S reference sequences (derived from the RDP 16S Training Set and supplemented by sequences from the RefSeq 16S set) using: (i) the RDP classifier to assign a taxonomy, and (ii) the usearch_global command (Edgar 2010) to find the closest match to the each OTU in the reference set.
For each mOTU, the number of non-rarefied reads per sample post-filtering is reported. 

 

 

Files

-Unfiltered demultiplexed 16S data: These fastq files contain demultiplexed unfiltered sequencing data. Two fastq files per sample resulting from paired-end sequencing ('R1' and 'R2'). 16S Prokaryote amplicons were amplified with primers 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’) (Parada et al. 2016; Apprill et al. 2015). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Demultiplexing of original sequencing files was performed using the GHAP pipeline (Greenfield 2017; available at https://doi.org/10.4225/08/59f98560eba25).

-Unfiltered demultiplexed 18S data: These fastq files contain demultiplexed unfiltered sequencing data. Two fastq files per sample resulting from paired-end sequencing ('R1' and 'R2'). 18S Eukaryote amplicons were amplified with primers All18SF (5’-GGTGCATGGCCGTTCTTAGT-3’) and All18SR (5’-CATCTAAGGGCATCACAGACC-3') (Hardy et al. 2010). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Demultiplexing of original sequencing files was performed using the GHAP pipeline (Greenfield 2017; available at https://doi.org/10.4225/08/59f98560eba25).

-Unfiltered demultiplexed COI data: These fastq files contain demultiplexed unfiltered sequencing data. Two fastq files per sample resulting from paired-end sequencing ('R1' and 'R2').  COI Metazoa amplicons were amplified with primers mlCOIintF (5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’) and jgHCO2198 (5’-TAIACYTCIGGRTGICCRAARAAYCA-3’) (Leray et al. 2013). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Demultiplexing of original sequencing files was performed using the GHAP pipeline (Greenfield 2017; available at https://doi.org/10.4225/08/59f98560eba25).

-Filtered 16S data: This file contains the filtered mOTUs x samples table for the 16S Prokaryote dataset. Amplicons were amplified with primers 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’) (Parada et al. 2016; Apprill et al. 2015). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Data were filtered according to the filtering procedure described in Pansu et al. (2021): ‘Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota’ (Marine & Freshwater Research). For each mOTU, the number of reads per sample post-filtering is reported.

-Filtered 18S data: This file contains the filtered mOTUs x samples table for the 18S Eukaryote dataset. Amplicons were amplified with primers All18SF (5’-GGTGCATGGCCGTTCTTAGT-3’) and All18SR (5’-CATCTAAGGGCATCACAGACC-3') (Hardy et al. 2010). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Data were filtered according to the filtering procedure described in Pansu et al. (2021): ‘Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota’ (Marine & Freshwater Research). For each mOTU, the number of reads per sample post-filtering is reported.

-Filtered COI data: This file contains the filtered mOTUs x samples table for the COI Metazoa dataset. Amplicons were amplified with primers mlCOIintF (5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’) and jgHCO2198 (5’-TAIACYTCIGGRTGICCRAARAAYCA-3’) (Leray et al. 2013). Sequences were obtained by a 2 x 250 bp paired-end sequencing on Illumina MiSeq 2500 platform. Data were filtered according to the filtering procedure described in Pansu et al. (2021): ‘Comparison of an extracellular vs. total DNA extraction approach for environmental DNA-based monitoring of sediment biota’ (Marine & Freshwater Research). For each mOTU, the number of reads per sample post-filtering is reported.