Rhizospheric bacterial community composition
DNA was extracted from 0.5 g of soil using Nucleospin Soil Kit (Macherey
Nagel) with buffer SL1 and SX according to manufacturers’ instructions.
Amplicon sequencing of the V4 hypervariable region of the 16S rRNA gene
was performed on a MiSeq Illumina instrument (MiSeq Reagent Kit v3 (600
Cycle); Illumina, San Diego, CA, USA) using the universal eubacterial
primers 515F (Parada, Needham & Fuhrman 2016) and 806R […],
extended with sequencing adapters to match Illumina indexing primers. To
identify potential contamination during DNA extraction and
amplification, both extraction and PCR no template control samples were
performed. PCR was done using NEBNext high fidelity polymerase (New
England Biolabs, Ipswich, USA) in a total volume of 25 µl (5 ng DNA
template, 12.5 µl polymerase, 5 pmol of each primer, 2.5 µl 3% BSA).
PCR conditions were 5 min at 98 ˚C; 25 cycles of 10 s at 98 ˚C, 30 s at
55 ˚C, 30 s at 72 ˚C; 5 min 72 ˚C. PCR products were purified using
MagSi NGSprep Plus beads (Steinbrenner, Wiesenbach, Germany) and
quantified via PicoGreen assay. Subsequently, indexing PCR was performed
using the Nextera XT Index Kit v2 (Illumina, Inc. San Diego, CA, US) in
a total volume of 25 µl (10 ng DNA template, 12.5 µl NEBNext high
fidelity polymerase, 2.5 µl of each indexing primer) and the following
PCR conditions: 30s at 98 ˚C; 8 cycles of 10 s at 98 ˚C, 30s at 55 ˚C,
30s at 72 ˚C; 5min 72 ˚C. Indexing PCR products were purified using
MagSi NGSprep Plus beads, qualified and quantified via a Fragment
Analyzer™ instrument (Advanced Analytical Technologies, Inc., Ankeny,
USA), and pooled in an equimolar ratio of 4nM.
Sequences were analyzed on the Galaxy web platform ( Afgan et al, 2016).
FASTQ files were trimmed with a minimum read length of 50 using Cutadapt
(Martin 2011). Quality control was performed via FastQC (Andrews 2010).
For subsequent data analysis DADA2 pipeline (Galaxy Version 1.20)
(Callahan et al., 2016) was used with the following trimming and
filtering parameters: 20 bp were removed n-terminally, and reads were
truncated at position 240 (forward) and 180 (reverse), respectively,
with an expected error of 3 (forward) and 5 (reverse). The resulting
unique amplicon sequence variants (ASV) were assigned to the SILVA
v138.1 (release 99%) reference database. To exclude potential
contamination, ASV occurring in no template controls, as well as
unassigned, mitochondrial and chloroplast, reads, and singletons (ASV
represented by only one read) were removed from the dataset, resulting
in an average read loss of 5.2%. To compare samples without statistical
bias, 38724 reads were subsampled in all samples, reflecting the lowest
observed read number. Rarefaction analysis indicated that this sampling
depth was sufficient for further analysis of samples (Supplementary
Figure. 1). The sequence data obtained in this study are deposited in
the short-read archive of NCBI under accession number PRNJA989406
(https://dataview.ncbi.nlm.nih.gov/object/PRJNA989406?reviewer=ficg6d69j4e0tit266actkpjd9).