Computational protocol: Assessing soil bacterial community and dynamics by integrated high-throughput absolute abundance quantification

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Protocol publication

[…] According to the method reported by , each composite DNA sample for sequencing was mixed by combining equal amounts of DNA extraction of the three replicates. The bacterial communities of the Cont, E9 to E4, P, W, and S treatments were amplified with the V4 region of primers (520F, 5′-AYTGGGYDTAAAGNG-3′ and 802R, 5′-TACNVGGGTATCTAATCC-3′), and then sequenced with Illumina Miseq platform following standard protocols. The quality control of raw sequencing reads was performed using QIIME software (version 1.7.0) (). The sequences were removed if the average quality in a moving 5 bps window lower than Q20, length less than 150 bps and having the ambiguous barcode. The paired-end sequences were spliced by the same index with overlap no less than 10 bps and no mismatch using FLASH (). The chimera sequences were de novo identified and removed using UCHIME ().After quality filtering, the sequences clustering was performed using UCLUST with a 97% similarity and sorted out as operational taxonomic units (OTUs) (). Then, the OTUs were classified using RDP-classifier to match to SILVA database release 119 (; ). The raw data obtained in this research were deposited to NCBI SRA (Sequence Read Archive; http://www.ncbi.nlm.nih.gov/sra/) under accession numbers SRP097773 and SRP105351. [...] The absolute abundance of each phylum or genus in the soil bacterial community was calculated by multiplying total bacterial quantities (the copy number of 16S rRNA gene of total bacteria from qPCR) and the corresponding relative abundance from high-throughput sequencing (). The calculated absolute abundances of Escherichia-Shigella, Esch-V3 and Esch-V4, were based on its relative abundance and the total bacterial quantities measured by V3 and V4 regions of 16S rRNA gene, respectively. Since the 16S rRNA gene varied in copy numbers among the different bacteria genomes (; ), seven copies of 16S rRNA gene and 1 copy of fliC gene in one genome of strain EDL993 (NZ_CP008957, NCBI: http://www.ncbi.nlm.nih.gov) were considered in this study. The absolute abundances of Escherichia-Shigella (Esch-V3 and Esch-V4) and 7 × fliC gene copies were logarithmically transformed before performing linear regression analysis.Linear regression was performed using Origin 2016 (OriginLab, Northampton, MA, USA), and analysis of variance (ANOVA) used with SPSS 20 (IBM, Armonk, USA). The homogeneity of variances was tested by Levene’s test before ANOVA. Tukey’s honestly significant difference test was used when the variances were homogeneous. Otherwise Tamhane’s T2 test was used. Heatmaps and cluster analyses were performed using R software version 3.3.1 with pheatmap package (; ). […]

Pipeline specifications

Software tools QIIME, UCHIME, UCLUST, RDP Classifier, PHeatmaps
Databases SRA
Applications 16S rRNA-seq analysis, Transcriptome data visualization
Organisms Bacteria