They were grown in a warm summer with continental or hemiboreal climate, and the recorded climate profiles Additional file 1: Figure S1 , which started 2 weeks before sampling, show relatively warm temperatures, moderate but fluctuating precipitation, sunshine and atmospheric pressure levels, overall low wind levels, and average air humidity. In addition to the phyllo- and rhizosphere samples, bulk soil was sampled for metagenome sequencing.
All samples were stored on ice and immediately processed after arrival at a nearby laboratory. The microbial fractions were extracted separately for the rhizo- and phyllospere, and three independent single replicates per habitat, consisting of 15—20 leaves or roots, respectively, were collected and stored separately.
For the homogenization of the samples, 5 g of plant material per replicate was physically disrupted with a sterile pestle and mortar, re-suspended in 10 ml of 0. The nested PCR was carried out as described by Heuer et al.
Chimeras and remaining sequences of non-target, plastidal, and mitochondrial origin were removed. Unique reads were classified with the RDP classifier [63] v2. Centiscape v. Five grams of each sample phyllo-, rhizosphere, and bulk soil were weighed, transferred into sterile plastic bags together with 10 ml 0. Nom, France. Samples were placed at interims of 5 min on ice. Homogenized cell suspensions were further transferred into S34 tubes, and centrifuged at 10, rpm for 20 min.
For each of the datasets, only high-quality reads were retained for further processing, resulting in the following read numbers to process further: phyllosphere: 41,, Reads were aligned to the reference genomes of Brassica oleracea BOL; ncbi. To align the quality-filtered reads to the reference genomes, Bowtie 2 [39] was used, with the default alignment parameters. Additional file 1: Table S2 shows the alignment results.
Those reads that aligned to at least one of the three genomes were excluded from further processing. Thus, only those reads that were not aligned to any of the three reference genomes were used as input to the assembly program. The phyllosphere and soil samples had the largest and smallest numbers of aligned reads among the three samples, respectively, which was to be expected. Assembly of the reads into contigs was performed using the Velvet version 1.
Since the choice of the k-mer length is crucial, multiple assemblies with different k values were performed on each sample in order to determine the best k-mer length. In all assemblies, the expected coverage value was automatically found by Velvet and the insert length for paired-end reads was set at Additional file 1: Table S3 shows the key statistics of the final assemblies, where k is the length of the k-mer used.
For taxonomic analyses, the blastn program was run on all the filtered contigs on the TimeLogic DeCypher boards www. The blastn results from each sample were imported into MEGAN to perform the taxonomy analysis for the sample, where each contig is assigned to a bacterial lineage at the lowest rank possible with sufficient confidence.
An identical number of contigs assigned to bacteria the smallest such number among the phyllosphere, rhizosphere and bulk soil samples were randomly extracted from each sample and used to compare subsequent bacterial taxonomic and functional analyses results among the three samples.
Therefore, the taxonomy and functional analyses of the bacteria are based on the blastn and blastx results, respectively, for the 89, subsampled contigs from each sample. Similarly, the taxonomy and functional analyses of Enterobacteriaceae are based on the blastn and blastx results, respectively, for the subsampled contigs from the phyllosphere and rhizosphere samples, respectively.
A very low number of contigs from the soil sample was assigned to the Enterobacteriaceae; therefore, we were not able to include them in a meaningful comparison. The antibiotic resistance protein sequences for metagenomic data were downloaded from the CARD website card. A single hit of a contig to an antibiotic resistance protein was counted as multiple antibiotic resistance category hits, if the protein mapped to multiple categories.
Abundances within the enterobacterial fraction were analyzed in a separate bioinformatics approach based on non-assembled metagenome sequences. The processing included the removal of artificial replicate sequences [29], low-quality sequences [21], short sequences, and sequences containing ambiguous bases.
For the isolation of enterobacteria, homogenized cell suspensions from the second sampling were plated on growth media. In total, aerobic bacteria were isolated from the arugula phyllosphere and rhizosphere as well as bulk soil.
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