Computational protocol: Geographic distribution of cadmium and its interaction with the microbial community in the Longjiang River: risk evaluation after a shocking pollution accident

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Protocol publication

[…] Paired-end reads of the original DNA fragments from high throughput sequencing were merged using FLASH and assigned to each sample according to their unique barcodes. The 16S rRNA genes were processed and analysed using the open-source software QIIME, , and in-house Perl scripts were used to analyse alpha (within samples) and beta (among samples) diversity. First, sequence reads were filtered (fastq maxee = 0.5 and fastq trunclen = 289), replication was removed, and singletons were discarded. The Chimera Slayer (CS) tool was used for chimaera detection. Then, the CD-HIT package and the QIIME script “pick_de_novo_otus.py” were used to identify OTUs by making a OTU table, and sequences with ≥97% similarity were assigned to the same OTUs. Representative sequences for each OTU were selected, and the RDP classifier was used to annotate taxonomic information for each representative sequence. To compute the alpha diversity, the OTU table was rarefied, and two metrics were calculated: Chao1 estimates microbe abundance, and the Shannon index is used to estimate the number of unique OTUs found in each sample. Rarefaction analysis was used to quantify the representativeness of the sequencing dataset. Hierarchical cluster analysis was carried out using Bray–Curtis similarity based on the abundance of all OTUs in the stats package of R. […]

Pipeline specifications

Software tools QIIME, CD-HIT, RDP Classifier
Application 16S rRNA-seq analysis
Chemicals Cadmium