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

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.

监测沉积物生物群是评估水生生态系统质量的关键步骤。基于环境DNA的生物监测方法越来越受到青睐；然而，从沉积物中提取总DNA的商业化试剂盒和方案仍然价格昂贵且耗时。此外，它们只能容纳少量沉积物，可能无法充分代表当地生物多样性，尤其是对于大型生物。在本研究中，我们评估了一种成本效益高、耗时低的细胞外DNA提取方法的可靠性，该方法能够处理大量起始材料，用于表征三种沉积环境中的细菌、真核生物和动物群落。采用细胞外方法提取的DNA浓度至少与商业试剂盒获得的浓度相似。局部多样性估计并未偏向任何特定的提取方法，尽管根据沉积物类型观察到特定的反应。群落组成和β多样性模式受到提取方法和起始材料初始量的适度影响；差异对于大型生物比微生物更为重要。因此，细胞外DNA方法在生物监测沉积群落方面显示出与基于商业化试剂盒的方法相当的鲁棒性和效率。其相对较低的成本和更快的处理时间使其成为大规模水生环境生态评估的有前景的替代方案。