From high throughput 454 GS FLX data analysis process of 16S RNA gene sequences using barcoding to bacterial community exploration

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(11) Fromhighthroughput454GSFLXdataanalysisprocessof16SRNA gene sequences using barcoding tobacterialcommunityexploration genesequencesusingbarcoding to bacterial community exploration L. Cauquil1,, J. Mariette2, C.Rousseau3, A. Zened1, F. Enjalbert1, A. Troegeler, 1 C. Klopp2 and S. Combes1 1UMR. 1289 Tandem, INRA / INPT-ENSAT / ENVT, Castanet-Tolosan, France 2Plateforme bio-informatique Genotoul, Biométrie et Intelligence Artificielle, INRA, Castanet-Tolosan, France, 3INPT-ENSAT, Castanet-Tolosan, France Laurent.Cauquil@toulouse.inra.fr. Since sequences might be randomly over represented within a run when sequencing is done using the Roche 454 pyrosequencing plateform (Gomez-Alvarez et al. 2009), random nucleotide dimeres were introduced in the sequence.. The bacterial community of 97 cow rumen samples was explored using a 440 bp V3-V4 16S RNA amplicon. E h sample Each l is i barcoded b d d using i a couple of MIDs.. 0. MID1. NN. 350. 800. 1500. V3. V4. NN. Primer Rev. Primer Forw. Pipeline. Eigth beads instead of one. MID2. Rawdata :889029reads Reads were grouped into Operation Taxonomic Unit (OTU) using Mothur software (http://www.mothur.org) according to a fixed cutoff of 0.1. For each OTU the dimere distribution was compared to the run margin using a chi-square test.. 16Spyrocleaner(python) Length >150nt Sequence with oneNareremoved Homopolymere H l >10areremoved 10 d. The distribution was considered to be biaised from the margin if a global chi-square was higher than 24.996 (ddl=15, p-value=0.05). Each dimere distribution was then tested and sequences were suppressed until an acceptable chi-square value.. 19.7%. Taxonomy levels. MOTHUR(http://www.mothur.org) Alignment (Silvabase) Distancematrix Assigning sequences toOTU. Groups identified. %Affiliation. Phylum. 15. 100. Class Order. 24 51. 98 95. Family Genus. 114 293. 84 50. 1.4% of reads not affiliated to any phylum were removed.. NNpyrocleaner (python) Remove overrepresented sequences (0.24%) Taxonomic assignation (bootstrap 60%). Actinobacteria Bacteroidetes Firmicutes Proteobacteria. MOTHUR(http://www.mothur.org). Others. Alignment Alignment Badaligned seq were removed (1%) Distancematrix Assigning sequences toOTU. 706155reads 7249reads/sample. 868OTU/sample (cutoff 0.1) Coverage of95.9%. Diversity indices. Mean distribution of major bacterial groups at the phylum level. Analysis. 0 0 0 0 0 0 … 0. 0.10. … … … … … … … …. 0.05. 0 0 0 0 0 0 … 1. Standardise abundance. 57 53 4 92 25 49 … 75. Actinobacteria Bacteroidetes Fimicutes Proteobacteria. 0.00. 0 0 0 0 0 2 … 0. Taxon430. Taxon7. Taxon3 0 3 0 1 1 3 … 4. …. 0 0 0 0 0 2 … 0. Taxon9. 0 1 0 0 0 0 … 0. Taxon8. Ruminal fluid 1 Ruminal fluid 2 Ruminal fluid 3 Ruminal fluid 4 Ruminal fluid 5 Ruminal fluid 6 ... Ruminal fluid 97. Taxon2. X matrix. Taxon1. 0.15. From the 97 samples, we identified 430 different taxa leading to a taxonomic ecological matrix. The resulting data set can be used for ordination and nMDS/chord + clusters Ward/chord clustering analysis (after normalisation) using R software.. 0. 100. 200. 300. 400. Taxa. Plot of the mean of the standardise abundance of each taxa upon the 97 samples. Ordination plot (nMDS) of the 97 samples and classification in 4 clusters. Gomez-Alvarez V., Teal T.K. and Schmidt T.M. Systematic artifacts in metagenomes from complex microbial communities. ISME Journal (2009) 3, 1314–1317; doi:10.1038/ismej.2009.72.. ALIMENTATION. 4th Congress of European Microbiologists, FEMS 2011 Switzerland, June 26-30. AGRICULTURE ENVIRONNEMENT.

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