Computational protocol: Temporal dynamics of hot desert microbial communities reveal structural and functional responses to water input

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Protocol publication

[…] Metagenomic DNA was extracted from 0.25 g of soil using the PowerSoil®- htp 96-Well Soil DNA Isolation (MoBio, Carlsbad, CA, USA) following manufacturer’s instructions. In total, DNA was extracted from 128 samples according to the methods used by the Earth Microbiome Project (http://www.Earthmicrobiome.Org/emp-standard-protocols/dna-extraction-protocol/). Sample sequencing was performed using bacterial/archaeal 515f and 806r error-correcting barcoded primers as described previously. Illumina MiSeq sequence data was screened using the QIIME toolkit with the following parameters: quality score >25, sequence length >200 and <400, maximum homopolymer of 6, 0 maximum ambiguous bases, and 0 mismatched bases in the primers and barcodes. Sequence counts before and after quality filtering were 348687 and 124597, respectively. Following chimera removal (USEARCH 6.1), 115102 sequences remained. OTUs were picked at the 97% identity level using UCLUST with the default settings. Phylogenetic identification was performed against the most recent Greengenes database (gg_13_5). After removal of singletons, samples were rarefied to 328 reads, which represents the lowest number of sequences obtained for a single sample. In total 26 samples, which failed quality control, were excluded from further analysis, yielding a total of 102 samples. […]

Pipeline specifications

Software tools QIIME, USEARCH, UCLUST
Databases Greengenes EMP
Applications Phylogenetics, Metagenomic sequencing analysis
Chemicals Carbon Dioxide