Valentin Vasselon 1, Agnès Bouchez 1, Isabelle Domaizon 1, Maria Kahlert 2, Frédéric Rimet 1 Towards standardization of DNA extraction for next- generation biomonitoring with diatoms 1 INRA CARRTEL (Thonon-les-Bains, France) 2 Swedish University of agricultural Sciences (Uppsala, Sweden)

Two ways to get a diatom community composition Morphological Approach (microscope) Molecular Approach - HTS (High-Throughput Sequencing) Quality index (IPS, DI-CH,…) Quality assessment Taxonomic expertise (patterns recognition) Species inventory Taxonomic assignment of reads (reference database)

What can affect the molecular inventory? Bioinformatics Sampling DNA extraction Sequencing PCR amplification All these steps can create biases, need of tests for standardization Reference database Impact of the DNA extraction methods ? (Lazarevic et al. 2013, Staley et al. 2015) ?

Materiels and Methods - DNA extraction Mechanical (beads) Enzymatic (Rnase or Proteinase K) Thermal shock (-80°C, 55°C) Sonication (ultrasonic bath) Enzymatic (Proteinase K) MN-SoilMN-PlantS-PlantQ-BloodSA-Gen I. Cell lysis II. DNA purification Filtration column DNA fixation (silica column) III. DNA concentration Comparison of 5 DNA extraction methods (Nguyen et al. 2011, Eland et al. 2012) Centrifugation (pellet of contaminants) Centrifugation (pellet of DNA)

Materiels and Methods – Sampling sites Aire Edian Lake Pol10 Ref7 P M36 Lakes and streams 3 Geographical areas Physicochemical characteristics ([organic matter], presence of metals, …) ≠ diatom assemblages Tropical island (Mayotte) France Sweden

Sampling Sequencing PCR amplification DNA extraction Bioinformatics Rsyst::diatom (Rimet et al. 2016) Reference database ? Materiels and Methods – High-throughput Sequencing (HTS) MN-Soil MN-Plant S-Plant Q-Blood SA-Gen rbcL gene, 312 bp (Kermarrec et al. 2014) PGM ion torrent (INRA, Bordeaux, France) Mothur (Schloss et al. 2009)

Materiels and Methods – Comparison criteria  DNA quantity and purity DNA quantity - Picogreen quantification PCR inhibitors detection (qPCR)  Community inventories obtained from HTS Richness and diversity Community structure – NMDS  Comparison between molecular and morphological inventories Community composition Biotic diatom index (IPS)

Comparison of the 5 DNA extraction methods DNA quantity and purity SA- Gen = Highest efficiency Presence of inhibitors Highest inhibition for SA-Gen DNA quantity DNA purity Inhibition level ND – Not Determined (out of range values) PCR and sequencing were successful with all methods DNA extraction[DNA extracted] methodµg/g of biofilm MN-Soil106.7 MN-Plant140 S-Plant373.3 Q-Blood246.7 SA-Gen1386.7

DNA reads OTU1 OTU2 OTU3 OTU4 MN_Edian I_Edian Otu1000 Otu Otu3000 Otu4202 Otu5000 Otu OTU composition of samples Clustering according to site No effect of DNA extraction methods on inter-site comparison Comparison of the 5 DNA extraction methods Community inventories obtained from HTS Clustering in OTUs 95 % similarity Richness and Diversity No significant effect of extraction method on the OTU richness (Chao estimator) and diversity (Shannon) Community Structure NMDS plot of Bray-Curtis dissimilarity 81% (p < 0.001) 90% (p < 0.001) 86% (p < 0.001) 72% (p < 0.002) 91% (p < 0.004) 42% (p = 0.63) 61% (p = 0.25) 72% (p < 0.001) For 6 sites, major part of intra- site variability was explained by the DNA extraction methods Permanova results – effect of DNA extraction method Variance explained

Comparison of the 5 DNA extraction methods Community inventories obtained from HTS Origin of intra-site variability SA-Gen and MN-Soil methods presented the highest dissimilarity within sites Simper analysis - OTUs contributing to more than 1 % of dissimilarity Taxonomy was assigned to these OTUs using the molecular reference database (Rsyst::diatom) No difference was detected on the presence/absence of taxa

Comparison of the 5 DNA extraction methods Community inventories obtained from HTS Origin of intra-site variability SA-Gen and MN-Soil methods presented the highest dissimilarity within sites Simper analysis - OTUs contributing to more than 1 % of dissimilarity Taxonomy was assigned to these OTUs using the molecular reference database (Rsyst::diatom) No difference was detected on the presence/absence of taxa Variation in the proportion of specific taxa was observed GenusSA-GenMN-Soil Nitzschia Amphora Encyonema Gomphonema Navicula Read sum of OTUs contributors Diatom silica wall is resistant (Hamm et al. 2003) Variation of cell lysis efficiency ?

Good correspondence at Genus level Comparison of the 5 DNA extraction methods Molecular versus morphological inventories Correspondence between molecular and morphological inventories Only in molecular inventories Detected in both inventories Only in morphological inventories and absent from reference database

Good correspondence at Genus level Low correspondence at Species level due to incompleteness of the reference database Comparison of the 5 DNA extraction methods Molecular versus morphological inventories Correspondence between molecular and morphological inventories Only in molecular inventories Detected in both inventories Only in morphological inventories and absent from reference database

Comparison of the 5 DNA extraction methods Molecular versus morphological inventories Correspondence between molecular and morphological inventories Only in molecular inventories Detected in both inventories Only in morphological inventories and absent from reference database Site quality assessment using Biotic Index (IPS) France Sweden Tropical island (Mayotte) IPS values were not different between DNA extraction methods Molecular and Morphological IPS are congruent Except for the tropical island sites, for which the taxa are poorly referenced IPS

Conclusion The 5 DNA extraction methods are suitable for HTS metabarcoding The taxonomic composition of diatoms assemblages is not affected but the relative abundances are impacted (lysis bias) Quality assessment is not affected by the DNA extraction methods and is congruent with the morphological approach SA-Gen method is probably the best choice Cheapest (24 to 39 times) High quantity of DNA extracted No impact of PCR inhibitors on HTS Further work is required to standardize the full process Bioinformatic Sampling DNA extraction Sequencing PCR amplification

Thank you for your attention !