Computational protocol: Urea Amendment Decreases Microbial Diversity and Selects for Specific Nitrifying Strains in Eight Contrasting Agricultural Soils

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Protocol publication

[…] Sequence data were processed and analyzed using mothur ver. 1.35.1 (), and processed as described previously (). Briefly, samples were trimmed to 160 nt, paired-end joined using fastq-join software (), and trimmed for quality based on quality score (>35), homopolymer length (8 nt), ambiguous bases, and primer mismatches (2 nt). Samples were aligned against the SILVA database ver. 123 (), subjected to a 2% pre-cluster step to remove sequence errors (), and chimeras were identified and removed using UCHIME (). Operational taxonomic units (OTUs) were identified at 97% similarity using the complete-linkage algorithm and taxonomic classifications were made using the version 14 release from the Ribosomal Database Project ().Alpha diversity indices, as well as Good’s coverage, were calculated using the Shannon index and abundance-based coverage estimate (ACE) using mothur. Differences in beta diversity were evaluated using Bray–Curtis dissimilarity matrices (), which accounts for differences in abundance of OTUs. Differences in community composition were evaluated by analysis of similarity (ANOSIM) (), while differences in sample clustering were evaluated by analysis of molecular variance (AMOVA) (). Ordination of samples was performed by principal coordinate analysis (PCoA) (). Operational taxonomic units that differed significantly in abundance by treatment were determined using linear discriminant analysis of effect sizes (LEfSe) (), and were subsequently classified to families. Oligotyping analyses were used to determine potential species- and strain-level distributions among nitrifiers, according to recommended best practices (). An oligotype was defined as present in more than 1% of reads, present in at least three samples, the most abundant unique sequence occurred at a minimum of 50 reads, and if the minimum read abundance throughout the dataset was at least 250. […]

Pipeline specifications

Software tools mothur, ea-utils, UCHIME, LEfSe
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bacteria
Chemicals Nitrogen, Urea