Similar protocols

Pipeline publication

[…] end reads were merged with ea-utils. Following inspection of merged reads in FastQC all sequences were trimmed uniformly to 300 nucleotides leaving an average q-score of q37. Data was demultiplexed in Quantitative Insights Into Microbial Ecology (QIIME) requiring 95% of each read to have a minimum q-score of 20, and allowing no exceptions (-q 19 -r 0 -p 0.95). All demulitplexed sequence data were deposited to GenBank under BioProject number PRJNA397457 with sample accessions SAMN07458979-SAMN07459014. Demultiplexed data was screened for chimeras with VSEARCH using the usearch_ref option against the UNITE-based fungal chimera dataset, and subsequently screened for fungal ITS2 sequences with ITSx. Sequences were dereplicated on the first 100 bases with the prefix_suffix OTU picker in QIIME. OTUs were defined de novo with Swarm at a resolution of 1, and taxonomy was assigned using BLAST against the UNITE database. Reference sequences for C. posadasii and C. immitis were manually added to this database prior to taxonomic assignment. OTUs which represented <0.005% of the counts across the entire table were removed. The OTU table was rarefied to the lowest depth sample (2593 reads) or normalized by CSS or DESeq2 transformations. The rarefied table was used to explore differences in alpha-diversity using Chao1 estimator while all tables were used to explore differences in beta diversity or differential abundance of individual taxa using Bray-Curtis. Correlation between taxonomic identity and treatment groups was assessed with the Phi correlation from the R package ‘indicspecies’ at genus level. Correlation between relative abundance of Coccidioides spp. ITS2 amplicons and qPCR Ct values was assessed using a two-tailed Spearman rank correlation test., The 300 bp-amplicons representing OTUs specific to Coccidioides spp. were identified from each sampled location, and hereafter named denovo49 and […]

Pipeline specifications

Software tools ITSx, QIIME, DESeq2