Computational protocol: Exposure of Soil Microbial Communities to Chromium and Arsenic Alters Their Diversity and Structure

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Protocol publication

[…] Clone libraries were generated from the PCR amplicons of the ACR3 and arsB genes at each site. Clones were generated using a TOPO-TA clone kit (Invitrogen, Carlsbad, CA, USA) utilizing the optional blue/white screening test on LB media supplemented with ampicillin and S-Gal. Clones were randomly selected and checked for inserts by PCR prior to sequencing. Clone libraries, consisting of 48 clones for each gene and site, were replicated from stocks before being sequenced by Microgen (Microgen, Oklahoma City, OK). Sequencing was performed using an ABI 3730xl capillary sequencer (Applied Biosystems) Sequences underwent quality screening, primer removal, and binning into Shared Operational Taxonomic Units (OTUs) at a cutoff level of 90% with DNAstar (DNAStar inc., Madison, WI, USA). Quality OTUs were taxonomically identified using BLASTx (National Center for Biotechnology Information, Bethesda, MD, USA), and these results were combined with sequences identified in Cai et al. for phylogenetic comparison. Sequences with poor sequence alignment, no BLAST hits, or BLAST hits to genes that did not encode ACR3 or arsB were removed from downstream analyses. OTUs and known sequences were aligned using Muscle and then imported into MEGA 5 for construction of phylogenetic dendrograms using the Neighbor-Joining algorithm with the following parameters: Jukes-Cantor correction model for nucleotides and 1000 Bootstraps. [...] Raw pyrosequencing reads were binned by barcode, quality screened by using an average minimum quality score of twenty, and trimmed of primer sequence using the RDP pyrosequencing pipeline (http://pyro.cme.msu.edu/index.jsp). Sequence reads were then imported into the phylogenetic software package Mothur version 1.14.0. 64 bit (http://www.mothur.org) for OTU (Operational Taxonomic Unit) generation, diversity estimates and classification. Full descriptions of workflows can be found within the Mothur manual. Before alignment, sequences with more than eight homopolymer nucleotides and outside of our length requirement (140–180 bases) were removed. Within Mothur, sequences were aligned to the Silva core sequence set using the NAST algorithm . Sequences were then chimera checked with Chimera Slayer, sequence distances were calculated with no penalization for end gaps, OTUs were clustered using the furthest neighbor algorithm and were then classified within Mothur using Greengenes taxonomy and the Silva database. OTUs0.03 (Species level Operational Taxonomic Units) taxonomy was obtained by consensus using a cutoff of 60%. Alpha diversity metrics (Abundance-based coverage estimator (ACE) , Chao (estimates total species richness) , Shannon Index (calculates diversity within a sample) , rarefaction curves and rarified phylogenetic diversity) were also calculated with Mothur. In order to calculate beta diversity with Unifrac and Faith’s phylogenetic diversity , , a phylogenetic tree was generated with the program FastTree version 2.1 . To build the phylogenetic tree OTUs0.03 from each sample were aligned to the Silva database and lane masked. Unifrac analysis was performed using the Fast Unifrac web interface . Pearson correlation coefficients for environmental factors and diversity estimates were calculated in excel with the CORREL correlation function. Significance of the correlation was tested against a null Student’s t-test model using a two-tailed distribution to assess significance (P≤0.05) . Redundancy analysis (RDA) was performed in R (http://cran.r-project.org/) using the vegan package with normalized OTU abundance and environmental chemical data.Pyrosequencing data has been deposited at NCBI Sequence Read Archive accession number SRA026044. Clone library sequences from ACR3 and arsB have been deposited in GenBank under the following accession numbers ACR3 (JQ409060-JQ409108) and arsB (JQ608478–JQ608486). […]

Pipeline specifications

Software tools BLASTX, MUSCLE, MEGA, mothur, UniFrac, FastTree, Fast Unifrac, vegan
Databases MicroGen
Applications Phylogenetics, 16S rRNA-seq analysis, Nucleotide sequence alignment
Diseases Arsenic Poisoning
Chemicals Chromium