Computational protocol: Transgenic banana plants expressing Xanthomonas wilt resistance genes revealed a stable non-target bacterial colonization structure

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Protocol publication

[…] In a deep-sequencing approach, we focused on the colonization by Gammaproteobacteria. The hypervariable V4 region of the 16S rRNA gene was amplified in a nested PCR approach with the Gammaproteobacteria specific primer pair Gamma395f/Gamma871r and the universal primer pair 515F/806R according to Köberl et al.. PCR products of three independent reactions were pooled in equal volumes and purified by employing the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). Amplicon libraries were generated and sequenced by a paired-end approach using the Illumina MiSeq platform (LGC Genomics, Berlin, Germany). The nucleotide sequences are available in the European Nucleotide Archive (www.ebi.ac.uk/ena) under the BioProject accession number PRJEB9422.Data analysis was performed by employing the software package QIIME 1.7 and 1.8. Joined paired-end reads with more than three consecutive low quality base calls (Phred quality score ≤20) were truncated at the position where their quality began to drop, and only reads with >75% consecutive high quality base calls, without any ambiguous characters, and longer than 200 nucleotides in length were retained for further analyses. All quality sequences were adjusted in the same orientation and clustered into operational taxonomic units (OTUs) with uclust, using 3%, 5%, and 10% dissimilarity thresholds. From each OTU the most abundant sequence was selected as the representative one, and the taxonomy of the representative set was assigned with the uclust-based consensus taxonomy assigner using an 80% confidence threshold. The representative sequence set was aligned with PyNAST. Chimera check was performed with ChimeraSlayer and potentially chimeric sequences were discarded. OTU tables at the different dissimilarity levels were constructed, and OTUs not assigned to the class of Gammaproteobacteria as well as singletons were removed from the dataset. For alpha and beta diversity analyses, OTU tables were rarefied at 1,590 reads. Diversity indices Shannon, Chao1 and observed species were determined based on the normalized clustering data. Significant differences were calculated with PASW Statistics 18 (SPSS Inc., Chicago, IL, USA) using the independent samples t-test and the Tukey post hoc test. Beta diversity was analyzed based on weighted UniFrac distances and ten jackknife replicates of the total rarefied datasets. Statistical analyses were performed using the adonis test (p ≤ 0.05) with 999 permutations. […]

Pipeline specifications

Software tools QIIME, UCLUST, PyNAST, ChimeraSlayer
Application 16S rRNA-seq analysis
Organisms Musa acuminata