Bioinformatic tools for metagenomic data analysis
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1 Bioinformatic tools for metagenomic data analysis MEGAN - blast-based tool for exploring taxonomic content MG-RAST (SEED, FIG) - rapid annotation of metagenomic data, phylogenetic classification and metabolic reconstruction CAMERA (JCVI, Calit2, UCSD) - metagenomic data repository and blast server 1
2 Getting started Download MEGAN and metagenomic datasets BLAST files: 2
3 MEGAN (Metagenome analyzer) D.H. Huson et al., MEGAN analysis of metagenome data. Genome Research, 17: (2007). Software designed to analyze metagenomic datasets and assign reads to a known taxon. What organisms are present and what are their relative proportions? What types of types of genes are present? 3
4 Input files: - BLAST files; BLASTX, BLASTN or BLASTZ - NCBI-NR, NCBI-NT, NCBI-ENV-NR, NCBI-ENV-NT or genome specific databases Image from Huson et al., 2007 MEGAN uses NCBI taxonomy to summarize and order results. 4
5 MEGAN (Metagenomics analyzer) Lowest common ancestor (LCA) assignment algorithm: Filters set by user: - Min. Support for Taxa (default = 2) - Min. Score (default = 30.0) - Top Percentage (default = 10.0) - Win ( Winner ) Score (default = 0) 5
6 Comparison of metagenomic samples Single metagenomic sample 6
7 MEGAN (Metagenomics analyzer) Let s start playing with MEGAN! 7
8 MEGAN on startup screen: - will take a minute or so to load NCBI tree and taxonomy Default tree showing NCBI taxonomy 8
9 Step 1: Importing a BLAST file: - File Import. Choose import file type (e.g. BLASTX, BLASTN) 9
10 Step 2: Choosing MEGAN output format: - Full Dataset OR Summary (choose Full Dataset - greater control over filters) 10
11 Step 3: Importing and parsing the BLAST file: - Note: some taxa will remain unidentified! Summary of blast results 11
12 Step 4: Generating tree showing taxonomic distribution of reads: - Note: NCBI tree replaced by taxonomy of assigned reads Displays tree at the species level 12
13 Step 5: Setting filtering criteria - Options Min Support, Min Score, Top Percentage and Win Score 13
14 Step 6: Show the number of reads assigned to each taxa/node - Tree Show number of Reads Assigned 14
15 Step 7: Collapsing/expanding the tree to different taxonomic levels -Tree Collapse at Taxonomical Level - choose higher level to collapse, or lower level to expand 15
16 Step 8: Changing the vertical and horizontal view of the tree Expand Expand vertically horizontally Contract Contract vertically horizontally 16
17 Step 9: Taxon discovery rate - Window Chart Taxon Discovery Rate 17
18 Step 9: Exporting and saving MEGAN output - File Save As - File Export Image 18
19 Exercise: Comparing metagenomic samples using MEGAN 1) Import the BLASTX against NR file for the OMZ 20m dataset. **Note: this should be done already** (File name: OMZ_20m_allDNA.fna.5000VSnr.FF.b10.v10.c250.blastx) 2) Import the BLASTX against NR files for the OMZ 45m OR 60m dataset. (File name: OMZ_45m_allDNA.fna.5000VSnr.FF.b10.v10.c250.blastx OR OMZ_60m_allDNA.fna.5000VSnr.FF.b10.v10.c250.blastx) 3) Change the Min Score filter to a bitscore of 40 for each dataset. 4) Using the Compare tool (found under File--->Compare), select both datasets and choose the normalize over all reads option. 5) Collapse the resulting tree to the phylum level. Note any major differences in the representation of difference taxonomic groups. 19
20 MG-RAST MetaGenome Rapid Annotation using Subsystems Technology Login: CMORE Password: CMORE - Accurate and consistent annotations of metagenomic data (12-24 hours) - Currently accepts 454 and Sanger sequences - Automatic metabolic reconstruction - Phylogenetic classification of rrnas and proteins - Comparison tools 20
21 MG-RAST Subsystem a generalization of pathway as a collection of functional roles jointly involved in a biological process or complex * Submit fasta nucleotide sequences * Project name and description * Metadata (location, depth, altitude, time of collection, habitat) 21
22 CAMERA Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis The aim of this project is to serve the needs of the microbial ecology research community by creating a rich, distinctive data repository and a bioinformatics tools resource that will address many of the unique challenges of metagenomic analysis. 22
23 CAMERA Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis CAMERA requires registration: CMORE_2008 CMORE
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