How to manage large 16S datasets in QIIME?

Preparing QIIME Environment

• Ensure you have QIIME 2 installed on your system. You can follow the official QIIME 2 installation guide specific to your operating system.

 

• Activate your QIIME 2 environment using the terminal/command prompt: source activate qiime2-X.X, where X.X is your version number.

Importing Data

• Prepare your sequence files. Typically, you will have FASTQ files from Illumina sequencing platforms.

 

• Use the following command to import your data into QIIME: qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path .fastq --output-path demux.qza.

 

• If data is in a different format, refer to the QIIME 2 documentation for the corresponding import commands.

Demultiplexing and Quality Control

• Use the demux plugin to visualize the quality of your sequences: qiime demux summarize --i-data demux.qza --o-visualization demux.qzv. This step helps you decide on trimming and filtering criteria.

 

• Perform quality filtering using DADA2 or another desired method available in QIIME 2: qiime dada2 denoise-paired --i-demultiplexed-seqs demux.qza --p-trim-left-f --p-trim-left-r --p-trunc-len-f --p-trunc-len-r --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats stats.qza, where values are chosen based on your quality plots.

Feature Table and Taxonomy Assignment

• Visualize your feature table to understand the number and type of sequences retained post quality filtering: qiime feature-table summarize --i-table table.qza --o-visualization table.qzv.

 

• Assign taxonomy to your sequences using a pre-trained classifier: qiime feature-classifier classify-sklearn --i-classifier .qza --i-reads rep-seqs.qza --o-classification taxonomy.qza. Ensure that the classifier is appropriate for your region (e.g., 16S V3-V4).

Creating Visualizations

• Create taxonomic bar plots to visualize the microbial composition: qiime taxa barplot --i-table table.qza --i-taxonomy taxonomy.qza --m-metadata-file .tsv --o-visualization taxa-bar-plots.qzv.

 

• Generate other visual reports using QIIME's diverse set of plugins for different kinds of analyses like alpha and beta diversity.

Exporting Data for Further Analysis

• Convert the QIIME 2 artifacts to formats suitable for other bioinformatics or statistical analyses, for instance: qiime tools export --input-path table.qza --output-path exported-feature-table.

 

• Consider exporting representative sequences and taxonomy data for use in phylogenetic analyses or for compatibility with other software.

Backing Up Your Work

• Regularly save both QZA and QZV files, as these serve as documentation of your workflow and results.

 

• Consider setting up automated backups or use repositories like GitHub for version control and collaboration.