ITS of soil metagenome: manipulation of diversity via sterilization and inoculation of forest soil_dilution 2 at day 370
Microcosms (four replicates for four treatments in both soils; 4 x 4 x 2 = 32) were destructively sampled at five time points, day 0, day 13, day 76, day 188, and day 370. DNA was extracted using MoBio PowerSoil kit per the manufacturers instructions. The 16S rRNA gene was analyzed using a protocol similar to Nagel et al. (2016), in which the(Caporaso et al. (2010) protocol was modified for use with the IonTorrent sequencing platform (316 chip, University of Western Australia, School of Biomedical Sciences). After quantification with a Qubit fluorometer, 1 ng of DNA was used to amplify the ITS1f-ITS2-R region of the fungal ITS (ITS1-F: CTTGGTCATTTAGAGGAAGTAA and ITS2-R: GCTGCGTTCTTCATCGATGC). A 90:10 mixture of these gene-specific primers and gene-specific primers tagged with Ion Torrent-specific adaptors and barcodes was used to achieve maximum amplification of all samples within 18-20 cycles, which minimizes primer-dimer formation and allowed optimal downstream purification. One hundred amplicons per chip were diluted a final concentration of 15 pM. A coupled Ion OneTouch 2PGM (Life Technologies Corporation, Carlsbad, CA) was used to create and sequence templated Ion Sphere Particles, which returned roughly 8 Gb for the ITS region. Used R. Edgar's USEARCH v7 (Edgar, 2010) command 'fastq_strip_barcode_relabel2.py' to remove barcodes (e.g., python fastq_strip_barcode_relabel2.py $PWD/reads.fastq GTCTTGGTCATTTAGAGGAAGTAA $PWD/barcodes.fa Ex > reads2.fastq). Individual files for each sample were created with a for loop in python. (References: Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:24602461. https://doi.org/10.1093/bioinformatics/btq461; Nagel R, Traub RJ, Allcock RJN, et al (2016) Comparison of faecal microbiota in Blastocystis-positive and Blastocystis- negative irritable bowel syndrome patients. Microbiome 19. https://doi.org/10.1186/s40168-016-0191-0; Caporaso JG, Kuczynski J, Stombaugh J, et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7:335336. https://doi.org/10.1038/nmeth.f.303)