Qiime2

Overview

Quantitative Insights Into Microbial Ecology (QIIME) is an open-source bioinformatics pipeline for performing microbiome analysis from raw DNA sequencing data. QIIME is designed to take users from raw sequencing data generated on the Illumina or other platforms through publication-quality graphics and statistics. This includes demultiplexing and quality filtering, OTU picking, taxonomic assignment, and phylogenetic reconstruction, and diversity analyses and visualizations. QIIME has been applied to studies based on billions of sequences from tens of thousands of samples.

QIIME Features:

  • Automatically track your analyses with decentralized data provenance — no more guesswork on what commands were run!

  • Interactively explore your data with beautiful visualizations that provide new perspectives.

  • Easily share results with your team, even those members without QIIME 2 installed.

  • Plugin-based system — your favorite microbiome methods all in one place.

Notes:

ARCC currently does not monitor the current/latest versions of the software. If you require an update to a version please remember to put a request in and include any plugins you require.

  • Qiime2 does have a collection of plugins. The ones available can be seen by typing qiime from the command line once the module has been loaded, as shown below. If you require a plugin not listed please put a request into ARCC and we can explore how best to make this available.

  • We are still learning to what extent qiime2 is parallelized. At this moment we believe that it can only run on a single node. Some plugins can make use of multiple cores on that node, which can be found by reading the documentation relating to that plugin. Since there is no consistent syntax across the plugins on how to make use of this, if you can not work it out yourself please contact ARCC and we'll be happy to help.

  • If the usage of the software increases and the demand warrants the managing of the central reference database then ARCC is happy to discuss and explore.

Note on Plugins and Versions

You will notice that the available plugins are not all available in every qiime2 version. Plugins are open source, and developed by third-parties and thus not necessarily regularly updated when Qiime2 is updated. ARCC will try and see if a plugin will install when we update Qiime2, but in a lot of cases we are running into dependency issues where a plugin has been developed using older versions of libraries than that used by the latest Qiime version. Until the plugin developer releases a new version compatible with the latest version of Qiime there is nothing we can do.

Using

Use the module name qiime2 to discover versions available and to load the application.

Multicore

Out of the box, qiime2 does not automatically run in parallel, but some of the plugins/commands can be configured to use multiple cores.
One example is classify-sklearn which is a pre-fitted sklearn-based taxonomy classifier. This command has the --p-n-jobs option that allows multiple cores to be used.

Getting Help

There are a number of commands to get help from the command-line:

[@blog1 ~]$ module load qiime2/2023.5 [@blog1 ~]$ qiime Usage: qiime [OPTIONS] COMMAND [ARGS]... QIIME 2 command-line interface (q2cli) -------------------------------------- To get help with QIIME 2, visit https://qiime2.org. To enable tab completion in Bash, run the following command or add it to your .bashrc/.bash_profile: source tab-qiime To enable tab completion in ZSH, run the following commands or add them to your .zshrc: autoload -Uz compinit && compinit autoload bashcompinit && bashcompinit source tab-qiime Options: --version Show the version and exit. --help Show this message and exit. Commands: info Display information about current deployment. tools Tools for working with QIIME 2 files. dev Utilities for developers and advanced users. alignment Plugin for generating and manipulating alignments. composition Plugin for compositional data analysis. cutadapt Plugin for removing adapter sequences, primers, and other unwanted sequence from sequence data. dada2 Plugin for sequence quality control with DADA2. deblur Plugin for sequence quality control with Deblur. demux Plugin for demultiplexing & viewing sequence quality. diversity Plugin for exploring community diversity. diversity-lib Plugin for computing community diversity. emperor Plugin for ordination plotting with Emperor. feature-classifier Plugin for taxonomic classification. feature-table Plugin for working with sample by feature tables. fragment-insertion Plugin for extending phylogenies. gneiss Plugin for building compositional models. longitudinal Plugin for paired sample and time series analyses. metadata Plugin for working with Metadata. phylogeny Plugin for generating and manipulating phylogenies. quality-control Plugin for quality control of feature and sequence data. quality-filter Plugin for PHRED-based filtering and trimming. sample-classifier Plugin for machine learning prediction of sample metadata. taxa Plugin for working with feature taxonomy annotations. vsearch Plugin for clustering and dereplicating with vsearch.
[@blog1 ~]$ qiime info System versions Python version: 3.8.16 QIIME 2 release: 2023.5 QIIME 2 version: 2023.5.0 q2cli version: 2023.5.0 Installed plugins alignment: 2023.5.0 composition: 2023.5.0 cutadapt: 2023.5.0 dada2: 2023.5.0 deblur: 2023.5.0 demux: 2023.5.0 diversity: 2023.5.0 diversity-lib: 2023.5.0 emperor: 2023.5.0 feature-classifier: 2023.5.0 feature-table: 2023.5.0 fragment-insertion: 2023.5.0 gneiss: 2023.5.0 longitudinal: 2023.5.0 metadata: 2023.5.0 phylogeny: 2023.5.0 quality-control: 2023.5.0 quality-filter: 2023.5.0 sample-classifier: 2023.5.0 taxa: 2023.5.0 types: 2023.5.0 vsearch: 2023.5.0 Application config directory /home/salexan5/.config/q2cli Getting help To get help with QIIME 2, visit https://qiime2.org
[@blog1 ~]$ qiime alignment --help Usage: qiime alignment [OPTIONS] COMMAND [ARGS]... Description: This QIIME 2 plugin provides support for generating and manipulating sequence alignments. Plugin website: https://github.com/qiime2/q2-alignment Getting user support: Please post to the QIIME 2 forum for help with this plugin: https://forum.qiime2.org Options: --version Show the version and exit. --example-data PATH Write example data and exit. --citations Show citations and exit. --help Show this message and exit. Commands: mafft De novo multiple sequence alignment with MAFFT mafft-add Add sequences to multiple sequence alignment with MAFFT. mask Positional conservation and gap filtering. # Overview of 'fragment-insertion' plugin [@blog1 ~]$ qiime fragment-insertion --help Usage: qiime fragment-insertion [OPTIONS] COMMAND [ARGS]... Description: No description available. See plugin website: https://github.com/qiime2/q2-fragment-insertion Plugin website: https://github.com/qiime2/q2-fragment-insertion Getting user support: https://github.com/qiime2/q2-fragment-insertion/issues Options: --version Show the version and exit. --example-data PATH Write example data and exit. --citations Show citations and exit. --help Show this message and exit. Commands: classify-otus-experimental Experimental: Obtain taxonomic lineages, by finding closest OTU in reference phylogeny. filter-features Filter fragments in tree from table. sepp Insert fragment sequences using SEPP into reference phylogenies. # Overview of the 'fragment-insertion' specific 'sepp' command. # Notice the '--p-threads' option for multiple thread/core usage. [@blog1 ~]$ qiime fragment-insertion sepp --help Usage: qiime fragment-insertion sepp [OPTIONS] Perform fragment insertion of sequences using the SEPP algorithm. Inputs: --i-representative-sequences ARTIFACT FeatureData[Sequence] The sequences to insert into the reference tree. [required] --i-reference-database ARTIFACT SeppReferenceDatabase The reference database to insert the representative sequences into. [required] Parameters: --p-alignment-subset-size INTEGER Each placement subset is further broken into subsets of at most these many sequences and a separate HMM is trained on each subset. [default: 1000] --p-placement-subset-size INTEGER The tree is divided into subsets such that each subset includes at most these many subsets. The placement step places the fragment on only one subset, determined based on alignment scores. Further reading: https://github.com/smirarab/sepp/blob/master/tutorial/s epp-tutorial.md#sample-datasets-default-parameters. [default: 5000] --p-threads INTEGER The number of threads to use. [default: 1] --p-debug / --p-no-debug Collect additional run information to STDOUT for debugging. Temporary directories will not be removed if run fails. [default: False] Outputs: --o-tree ARTIFACT The tree with inserted feature data. Phylogeny[Rooted] [required] --o-placements ARTIFACT Placements Information about the feature placements within the reference tree. [required] Miscellaneous: --output-dir PATH Output unspecified results to a directory --verbose / --quiet Display verbose output to stdout and/or stderr during execution of this action. Or silence output if execution is successful (silence is golden). --example-data PATH Write example data and exit. --citations Show citations and exit. --help Show this message and exit.

Data Resources

On the WildIris cluster we have downloaded the data resources from: https://docs.qiime2.org/2022.8/data-resources/# and https://docs.qiime2.org/2023.2/tutorials/feature-classifier/

When you load the qiime2/<version> module, it sets a QIIME_DATA_RESOURCES environment variable, that can be used to view/access the data resources.

Issues

Bus Error

If within a job a step fails with a bus error, then our first suggestion will be to look at the amount of memory you have/haven’t allocated for you job. From working with users, the majority of the time it is caused by not requested enough memory for the size of data you’re trying to analyze.

Our wiki page here https://arccwiki.atlassian.net/wiki/spaces/DOCUMENTAT/pages/377454617 provides an introduction on how to define memory resources within a slurm job