Biodiversity and Evolution - Metabarcoding

Environmental Metagenomics: Metabarcoding and Microbes at Lake Gollin.

Authors: Fritz Lekschas, Annkatrin Bressin, Melanie Liedtke, Nina Kersten

Source code: https://github.com/flekschas/bio-div-metabarcording

Transcript

1. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Environmental Metagenomics: Metabarcoding and Microbes at Lake Gollin Biodiversity and Evolution M.Sc. Bioinformatics SS14
2. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Metagenomics Source: http://www.vaiomer.com/sites/default/files/Fig1%20Metagenomic%20workflow_2.png • study of genetic material recovered directly from environmental samples • to reveal the biodiversity of an ecosystem • samples: water, soil
3. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Material Samples: Source: http://www.dorfschule-gollin.de/wp-content/uploads/2014/03/Home-21. jpg • Lake Gollin (Schorfheide: April, May, June 2010) • Samples from 8 different locations • LSU gene as marker • 4-5-4 sequencing
4. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Lake zones Source: http://www.lakeaccess.org/ecology/lakeecologyprim9.html
5. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Aim / Goal • Biodiversity: ◦ number of fungal taxa • Dominance: ◦ relative proportion • Habitat / Season: ◦ distribution among habitat / season Source:http://www.mycolog.com/chapter11b.htm
6. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Expectations • Terrestrial zone: ◦ passively introduced via wind, rainwater, inflowing streams • Littoral zone: ◦ hotspot for all kinds of fungi • Pelagic zone: ◦ only specialized species Source: (1) Christian M. Wurzbacher et al. (2010) Fungi in lake ecosystems (2) Sébastien Monchy et al. (2011) Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing
7. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Methods
8. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Analysis Pipeline
9. First Approach: BLAST • Demultiplex Reads • Quality Control • Barcode, Primer removal & trimming • BLAST reads against SILVA • Taxonomy analysis BASH ➔ SFF to FASTQ ➔ FASTX Toolkit ➔ Trimmomatic ➔ Create BLAST DB ➔ BLAST (Subsamples) ➔ MEGAN ➔ R
10. Second Approach: OTU Generation • Same read preparation • OTU Clustering • Creating OTU table • Taxonomic classification with SILVA BASH ➔ SFF to FASTQ ➔ FASTX Toolkit ➔ Trimmomatic ➔ UPARSE ➔ SINA ➔ R
11. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Important parameters • Trimming: window min score of 15 • BLAST: 500 reads per month per habitat • MEGAN with SILVA taxonomy ◦ LCA: min support 5 and min score 50 • UPARSE: 97% sequence similarity • SINA: ◦ 70% sequence similarity and 10 reads support
12. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Results
13. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Biodiversity in Habitats (BLAST)
14. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal Biodiversity (BLAST)
15. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Dominance (BLAST)
16. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal Dominance (BLAST)
17. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Biodiversity in Habitats (OTUs)
18. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal Biodiversity (OTUs) Months Fungi species
19. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Dominance (OTUs)
20. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal Dominance (OTUs)
21. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Bacteria vs. Eukaryotes (Blast)
22. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Discussion
23. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Habitat specifity • similar at benthic • but overall wide variations • more specific classification at blast ◦ less reads mapped to fungal taxa ◦ max 40 reads per sample Discussion: Biodiversity
24. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal Biodiversity Discussion: Biodiversity
25. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Discussion: Dominance • Expect high percentage in: ◦ littoral, reed, benthic, biofilm • low percentage: ◦ pelagic, sediment, sed, plankton
26. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Discussion: Dominance Reltive domiance of fungi in eukarytes
27. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Discussion: Dominance
28. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Discussion • Only few fungi detected with Blast (SILVA) ◦ Same with SINA (online) ◦ Sub sampling may be problematic ◦ too specific classification • Bacteria found although eucaryotic primer • Pipeline (without MEGAN) at https://bitbucket.org/flekschas/biodivex2
29. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Look Out • OTU - Pipeline ◦ Similarity threshold ◦ NCBI database instead of SILVA • Blast - Pipeline ◦ SILVA LSU taxonomy good but could be better ◦ SILVA to NCBI mapping kind of old (2 years)
30. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Thank you! Questions?
31. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Appendix
32. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Dominance BLAST (Absolute)
33. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Dominance OTUs (Absolute)
34. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal dominance BLAST (Absolute)
35. 08.07.2014 Bressin, Kersten, Lekschas, Liedtke Seasonal dominance OTUs (Absolute)