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.