Analysis of the rhizosphere bacterial microbiota structure by metabarcoding
The DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) was used to extract DNA from roots and soil samples. The amplification of bacterial DNA was carried out by LGC genomics GmbH (Berlin, Germany) using the primers 799f and 1115r, and the amplicons were sequenced on an Illumina MiSeq instrument with 300bp paired-end reads.
Demultiplexing was conducted with Illumina bcl2fastq 2.17.1.14 software following the clipping of barcode and sequencing adapters. Primers were removed using Cutadapt v3.0 (Martin 2011). Sequences were processed in R 4.1 with dada2 version 1.22.0 (Callahan et al. 2016). Due to adapter ligation-based library prep, the raw sequences were in mixed orientation. To get the correct final orientation for learning error rates, reads were split into two groups (forwardRead.forwardPrimer - reverseRead.reversePrimer, and reverseRead.forwardPrimer - forwardRead.reversePrimer), denoised separately and merged after chimera removal. Forward and reverse reads were truncated at positions 265 and 210, resulting in 4073 unique Amplicon sequencing variants (ASV). Taxonomic classification was performed using the q2-feature-classifier plugin from Qiime2 version 2021.8.0 with a naïve Bayes classifier trained on the Silva 138.1 NR99 database.