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Aalborg Universitet Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters Kirkegaard, Rasmus Hansen; McIlroy, Simon Jon; Larsen, Poul; Karst, Søren Michael; Albertsen, Mads; Nielsen, Per Halkjær Publication date: 2014 Document Version Early version, also known as pre-print Link to publication from Aalborg University Citation for published version (APA): Kirkegaard, R. H., McIlroy, S. J., Larsen, P., Karst, S. M., Albertsen, M., & Nielsen, P. H. (2014). Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters. Poster session presented at 15th International Symposium on Microbial Ecology, Seoul, Korea, Republic of. 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Downloaded from vbn.aau.dk on: September 17, 2016 Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters Rasmus Kirkegaard, Simon McIlRoy, Poul Larsen, Søren M. Karst, Mads Albertsen, and Per H. Nielsen Center for Microbial Communities, Aalborg University, Denmark Conclusions Introduction • PCR independent validation is needed when conducting amplicon based studies! • Four times the standard bead beating is recommended (160 s) in order to capture the microorganisms with relatively tough cell walls. • The Sundberg et al (2013) primer set seems promising for capturing the overall community composition of both bacteria and archaea. • Every step of the protocol introduces variance, particularly the DNA extraction. However, the workflow gives good reproducibility. To understand the ecology network in anaerobic digesters it is necessary to produce a representative overview of the microbial community. In this study we develop a method for reliable and reproducible identification and quantification of microorganisms involved in biogas production. We test the effect of changing the parameters in a DNA extraction dependent approach to community profiling. Methods Results Class level overview of the archaeal population extraction 10 kbp 6 kbp - Methanomicrobia - 3 kbp - Input (µL) Reactor temperature 500 50 Thermophilic Viborg Aalborg West - 1 kbp - Thermoprotei Thermoplasmata - Standard 6 FastDNA® SPIN kit for Soil Effect of bead beating on DNA yield. All DNA extractions were done with 50 µL of AD sludge as input using the FastDNA® SPIN kit for Soil. The standard bead beating is 40 s. Class level overview of the bacterial population 1.0 400 160 80 40 Effect of bead beating on DNA integrity. At high bead beating durations the DNA is fragmented. primers 16S rRNA gene 400 Optimal 160 80 MBGB 40 400 160 80 40 Intensity (ms-1) Mean frequency of most common residue in 50 bp window Misc Crenarchaeotal Group - Kit standard Duration (s) Mesophilic Methanocbacteria Thaumarchaeota Bead beating Effect of primer set on the observed community structure. DNA extractions were done with 160 s. All results are relative abundance (%). The V3-V4 (Sundberg et al (2013)) primer set seems to cover the archaeal diversity seen with cDNA sequencing while the archaea specific V4-V6 (Klindworth et al (2013)) primers changes the composition dramatically. Note that the cDNA sequencing and 16S amplicon results are not directly comparable, but can be used as a rough guideline. Genus level overview of the archaeal population Chlostridia 0.8 V7 V1 0.6 V2 0 V3 V4 V5 V6 500 Bacterioda V8 V9 Ashelford K E et al. 2005 1000 1500 Base position V4-6: 519F + 1049R (≈522 bp) V1-3: 27F + 534R (≈506 bp) Bacteria only (Lane 1991, Muyzer et al 1993) V3-4: 341F + 806R (≈465 bp) Archaea only (Klindworth et al 2013) Archaea and Bacteria (Sundberg et al 2013) f__Methanobacteriaceae Actinobacteria Betaproteobacteria Methanolinea Anaerolineae Alphaproteobacteria Deltaproeobacteria Methanosaeta NA Acidimicrobia Methanosarcina Sphingobacteria replication NA Methanospirillium Spirochaetes Thermomicrobia Sampling AAU Extraction Sample prep Sequencing Bioinformatic s Gammaproteobacteria Methanobrevibacter Bacili Synergistia WWE1 5 biological replicates Min. 3 extraction replicates 3 sample prep replicates 3 sequencing replicates >50,000 overlapping 300 bp paired end reads pr. sample Brachyspirae Thermophilic Mesophilic Aalborg West Viborg NA OP8 Thermotogae validation Leptospirae Cloacamonae PCR independent assessment using Illumina TruSEQ shotgun sequencing Planctomycetia Flavobacteria Total RNA extraction cDNA synthesis DNA sequencing Mapping to references [email protected] Effect of primer set on the observed community structure. DNA extractions were done with 160 s. All results are relative abundance (%). The V3-V4 (Sundberg et al (2013)) primer set seems to cover the diversity of the bacterial domain seen with cDNA sequencing. The V1-V3 amplicon illustrates that the entire amplicon workflow has a great level of reproducibility. www.cmc.aau.dk Fluorescence in situ hybridisation with archaea specific probes supports the amplcons. Methanosarcina is commonly observed in high abundance in thermophilic anaerobic digesters, typically forming microcolonies, while Methanosaeta are often present in abundance in mesophilic reactors as short filaments. Amplicon and FISH analyses of the thermophilic and mesophilic digester samples is consistent with these observations.