LifeWatch Scientific Community Meeting, Rome, 27-29 May 2019 NEXT GENERATION SEQUENCING: OPPORTUNITIES AND CHALLENGES OF A DISRUPTIVE TECHNOLOGY FOR BIODIVERSITY ASSESSMENT AND MONITORING Pedro Beja CIBIO – Research Centre in Biodiversity and Genetic Resources, University of Porto & ISA/University of Lisbon CONTEXT: HOT, CROWDED AND FLAT WORLD OPPORTUNITIES: NEW CONCEPTS, TOOLS AND APPROACHES BARCODING AND METABARCODING ARE PROMISING TOOLS EPHEMEROPTERA PLECOPTERA ORTHOPTERA MUSEUM COLLECTIONS ODONATA FIELD TRICHOPTERA LEPIDOPTERA SAMPLING Expert Morphological Stored Specimen taxonomists ID Voucher Online DNA sequencing Sample (leg) 96% ethanol Database > 6,200 specimens (4,900 identified to species level) > ~2200 species; 240 families Borkhausenia crimnodes : native Aproaerema sp. Undescribed from Argentina, first record in species; 1 individual barcoded; Europe detected in bat faeces. ♀ ♂ Isotrias penedana: undescribed Cryptic diversity: possible new species or divergent lineages (3.6% to 13.4%) female detected with barcoding Automated identification of multiple organisms from a single (mixed) sample (e.g., faeces, soil, water or bulk samples) containing: • cellular DNA (living cells/organisms) • extracellular DNA (dead cells/organisms) EXTRACTION HIGH-THROUGHPUT TAXON SEQUENCING IDENTIFICATION “MULTI-SPECIES” SAMPLE PCR BIOINFORMATIC ECOLOGICAL AMPLIFICATION DATA PROCESSING ANALYSIS What is the ecological “quality” of ecosystems and how is it affected by environmental and anthropogenic drivers? Pawlowski et al., 2018, Science of the Total Environment Metabarcoding directly from preservative ethanol to obtain the species composition of the bulk sample while keeping it for further investigation (Martins et al., 2019, Molecular Ecology Resources) PRESERVATIVE ETHANOL SAMPLES BULK SAMPLES 5 sites Detection of taxa targeted by the Water Framewor Directive, taxa suitable to build ecological quality indices (Martins et al., 2019, Molecular Ecology Resources) Variation in species composition among sites was large compared to variation associated with subsampling day, extraction method, extraction replicate and PCR replicate Source of variation df MS Rsq F P Site 4 6.12 0.38 62.35 0.0001 Site:Day 5 0.24 0.02 2.48 0.0001 Site:Day:Method 10 0.19 0.03 1.92 0.0001 Site:Day:Method:Extra 10 0.09 0.01 0.91 0.800 ction Site:Day:Method: 50 0.07 0.06 1.000 Extraction: PCR Residuals 338 0.1 0.51 Total 417 Using several markers (18S, 16S, COI) allows taxonomic coverage from diatoms to vertebrates in the same sample. What is the role of neutral processes, environmental filtering, biotic interactions, response traits and phylogeny in community assembly? Ovaskainen et al. 2017. Ecology Letters, 20, 561-576. Study Area: • Tua Valley Regional Park 70 sampling sites • Vineyards • Olive groves • Cork-oak woodlands • Riparian galleries 1 light trap per site (sunset to sunrise) Sampling: • July, September 2017 • April-September 2018 All sites sampled in 6 days per month (No taxonomic knowledge required) PCR DNA Bioinformatics Illumina DNA sequencing Invertebrate powder (COI, 420 bp) extraction 140 samples processed Other (14) (July and September 2017) Trichoptera 103 • 5.4 Million usable reads 22 Neuroptera • 9500 unique haplotypes 31 • ~1250 unique Arthropod taxa: Orthoptera • ~50% identified to Species 95 Lepidoptera • ~5% to Genus Hemiptera 430 • ~34% to Family 144 • ~8% to Order • ~2% to Class Coleoptera 163 Diptera 239 Total species richness Exclusive species Fourth corner analysis Moth Species assemblages How to predict structure, dynamics and responses to environmental change and ecosystem management? Raimundo et al. 2018. Trends in Ecology and Evolution. Study Area LTER Baixo Sabor (lter_eu_pt_002) Field Methods • Capture of individuals of all bat species (Spring/Summer 2016/17) • Mist-netting • Visits to roosts • Collection of faecal pellets Lab Methods • DNA Extraction, PCR, Metabarcoding • 1 COI marker for bat ID (202bp) • 2 COI markers for Prey ID (157bp; 205bp) 1280 pellets processed (20 bat species) • 24 Million usable reads Other • 1377 unique Arthropod taxa: Orthoptera 139 • ~60% identified to Species 49 Hymenoptera • ~10% only to Genus 53 Lepidoptera • ~15% only to Family Araneae 392 • ~12% only to Order 57 • ~2% only to Class Hemiptera • 4250 unique interactions 147 Coleoptera Diptera 159 354 Prey with frequency of occurrence > 10% 19% 14% 13% 12% Pine Processionary Crane Fly Turnip Moth Vine’s Rustic Moth Thaumetopoea pityocampa Tipula oleracea Agrotis segetum Hoplodrina ambigua 12% 12% 12% 10% 10% Green Lacewing Spittlebug Large Yellow Underwing Mayfly Moth Fly Chrysoperla sp. Neophilaenus sp. Noctua pronuba Caenidae sp1 Psychoda alternata Interaction Network Modularity Creating a Virtual Research Environment to enhance accuracy and reproducibility of NGS data for ecological applications Harnessing the power of miniaturisation to develop real-time sequencing devices for field deployment MinION https://nanoporetech.com/products/minion Connecting Earth Observation Systems with conventional or automated molecular monitoring and assessment of biodiversity Bohan et al., 2017, Trends in Ecology & Evolution THANKS FOR LISTENING!!.