Bug Fix: `evaluate-busco` Fails With Auto Lineage Selection

by Alex Johnson 60 views

Introduction

In this article, we will discuss a bug encountered while running the evaluate-busco command with the auto-select lineage option. This issue leads to a failure during the pplacer placement stage, which ultimately prevents the successful generation of BUSCO JSON output. We will delve into the specifics of the bug, how to reproduce it, the expected behavior, and the environments where this issue has been observed. This article aims to provide a comprehensive understanding of the problem and potential solutions or workarounds.

Understanding the evaluate-busco Command and BUSCO

Before diving into the specifics of the bug, let's briefly understand the context. The evaluate-busco command is a crucial tool in bioinformatics, particularly within the QIIME 2 ecosystem. It leverages BUSCO (Benchmarking Universal Single-Copy Orthologs) to assess the completeness of genome assemblies, metagenomes, and transcriptomes. BUSCO identifies the presence of a set of universal single-copy orthologs, which are genes expected to be present in all organisms within a specific lineage. By analyzing the presence and completeness of these genes, evaluate-busco provides valuable insights into the quality and comprehensiveness of the analyzed dataset.

The --p-auto-select-lineage option is a convenient feature that automatically selects the appropriate BUSCO lineage database based on the input data. This eliminates the need for manual selection, making the process more streamlined and user-friendly. However, as we will see, this option is implicated in the bug we are discussing.

The Bug: SEPP Failure During pplacer Placement

The core of the issue lies in the failure of SEPP (SATé-Enabled Phylogenetic Placement) during the pplacer placement stage. This failure occurs when evaluate-busco is run with the --p-auto-select-lineage option. The root cause is that pplacer, a phylogenetic placement tool, receives an incorrect directory specification. This leads to a fatal error, specifically a "Failure("Bad directory specification")" message. This error prevents the generation of crucial .jplace placement results, which are necessary for subsequent steps in the evaluate-busco pipeline.

The absence of .jplace files has a cascading effect. The q2-annotate plugin, which relies on these files, subsequently crashes with an IndexError. This is because it cannot find the expected BUSCO JSON output, which is generated based on the pplacer results. Therefore, the initial failure in pplacer placement effectively halts the entire evaluate-busco process.

This bug has been observed in multiple environments, including macOS and HPC Linux environments, indicating that it is not specific to a particular operating system. The consistent occurrence of the error when using --p-auto-select-lineage suggests that the issue is directly related to how the lineage is automatically selected and how the directory paths are handled in this mode.

Reproducing the Bug: Step-by-Step Guide

To reproduce this bug, you need to follow a specific set of steps. This allows developers and users to confirm the issue and test potential fixes. Here’s a detailed guide:

  1. Download the BUSCO Database: The first step is to download the complete BUSCO database. This database contains all available lineages, which can be quite large (approximately 500 GB in total). You can use the fetch-busco-db action, likely provided by the QIIME 2 environment or a similar bioinformatics tool suite, to download the database. Ensure that you have sufficient storage space before initiating the download.

  2. Run the evaluate-busco Command: Once the database is downloaded, you can execute the evaluate-busco command with the --p-auto-lineage option. Here’s an example command:

    mosh annotate evaluate-busco \
  --i-mags mags_derep.qza \
  --i-db busco_db.qza \
  --p-auto-lineage \
  --p-cpu 4 \
  --output-dir evaluate_busco_outdir \
  --verbose

    Let's break down this command:

    • mosh annotate evaluate-busco: This invokes the evaluate-busco command, likely within a specific environment or workflow management system.
    • --i-mags mags_derep.qza: This specifies the input metagenome-assembled genomes (MAGs) in a QIIME 2 artifact format (.qza). Replace mags_derep.qza with your actual input file.
    • --i-db busco_db.qza: This indicates the BUSCO database file, also in QIIME 2 artifact format. Replace busco_db.qza with the path to your downloaded BUSCO database.
    • --p-auto-lineage: This is the crucial parameter that triggers the bug. It instructs evaluate-busco to automatically select the lineage.
    • --p-cpu 4: This sets the number of CPU cores to use for parallel processing. Adjust this based on your system's capabilities.
    • --output-dir evaluate_busco_outdir: This specifies the output directory where the results will be stored. You can choose any suitable directory.
    • --verbose: This flag enables verbose output, which can be helpful for debugging.

  3. Observe the Error: After running the command, you should observe the error in the output. The error typically manifests as a failure during the pplacer placement stage, with the "Bad directory specification" message. You may also see the subsequent IndexError due to the missing BUSCO JSON output.

By following these steps, you can reliably reproduce the bug and verify any potential fixes or workarounds.

Expected Behavior vs. Actual Behavior

To fully appreciate the impact of this bug, it’s important to understand the expected behavior of evaluate-busco when using the auto lineage option. The expected behavior is that the command should run from start to finish without any issues. The auto lineage selection should correctly identify the appropriate BUSCO lineage database for the input data, and the pplacer placement should proceed smoothly, generating the necessary .jplace files. The subsequent steps, including the generation of BUSCO JSON output and the final results, should also complete without errors.

However, the actual behavior deviates significantly from this expectation. As we have seen, the pplacer placement fails due to an incorrect directory specification, leading to a cascade of errors that prevent the successful completion of the evaluate-busco command. This discrepancy between the expected and actual behavior highlights the severity of the bug and its potential impact on users who rely on this functionality.

Environments Where the Bug Occurs

This bug has been observed in a variety of environments, indicating that it is not tied to a specific operating system or hardware configuration. The reported environments include:

  • macOS: The bug has been reproduced on macOS systems, suggesting that it is not specific to Linux-based environments.
  • Ubuntu: Ubuntu, a popular Linux distribution, is another environment where this bug has been observed.
  • HPC Linux Environments: High-performance computing (HPC) environments, which are often used for large-scale bioinformatics analyses, are also susceptible to this bug. This is particularly concerning, as HPC environments are frequently used for computationally intensive tasks like metagenomic analysis.

The fact that this bug occurs across different operating systems and computing environments underscores its widespread impact and the need for a robust solution.

Impact of the Bug

The bug in evaluate-busco with the auto-select lineage option can have several significant impacts on users and their research:

  1. Inaccurate or Incomplete BUSCO Analysis: The primary impact is that the BUSCO analysis fails to complete successfully. This means that users cannot obtain accurate or complete assessments of the completeness and quality of their genome assemblies, metagenomes, or transcriptomes. This can lead to flawed conclusions and unreliable results.
  2. Wasted Computational Resources: Running evaluate-busco with the auto lineage option, especially on large datasets, can consume significant computational resources, including CPU time and memory. When the command fails due to this bug, these resources are effectively wasted, leading to inefficiencies and delays in research projects.
  3. Time and Effort: Troubleshooting and debugging this issue can be time-consuming and require significant effort. Users may need to spend considerable time trying to identify the cause of the failure and find workarounds. This can divert their attention from other important research tasks.
  4. Frustration and Reduced Productivity: Encountering unexpected errors and failures can be frustrating for users, particularly those who are less experienced with bioinformatics tools. This can lead to reduced productivity and a negative user experience.

Potential Causes and Workarounds

While the exact root cause of the bug may require further investigation by developers, we can speculate on some potential causes and explore possible workarounds.

Potential Causes

  1. Incorrect Directory Specification: The error message "Bad directory specification" suggests that the pplacer tool is receiving an invalid path to a required file or directory. This could be due to an issue in how the auto lineage selection process determines the correct path or how the path is passed to pplacer.
  2. Path Resolution Issues: Differences in path resolution between different operating systems (macOS, Linux) or environments (local machines, HPC clusters) could contribute to the problem. A path that is valid in one environment may not be valid in another.
  3. Version Incompatibilities: Incompatibilities between different versions of the software components involved (e.g., evaluate-busco, pplacer, BUSCO database) could also play a role. It's important to ensure that all components are compatible with each other.

Potential Workarounds

  1. Manual Lineage Selection: A possible workaround is to manually specify the BUSCO lineage using the --p-lineage option instead of using --p-auto-lineage. This requires the user to identify the appropriate lineage for their data, which may involve some manual investigation. However, it can bypass the bug associated with the auto lineage selection process.
  2. Check Directory Paths: Before running evaluate-busco, users can manually check the directory paths used by pplacer to ensure they are valid and accessible. This may involve examining the command-line arguments passed to pplacer and verifying the existence of the required files and directories.
  3. Update Software Components: Ensuring that all software components are up to date can sometimes resolve compatibility issues. Users should check for updates to evaluate-busco, pplacer, BUSCO database, and any other relevant software packages.
  4. Use a Consistent Environment: Running evaluate-busco in a consistent environment, such as a Conda environment or a Docker container, can help mitigate path resolution issues and version incompatibilities. This ensures that all dependencies are managed in a controlled manner.

Conclusion

The bug in evaluate-busco with the auto-select lineage option is a significant issue that can prevent users from obtaining accurate BUSCO analyses. The failure during pplacer placement, caused by a bad directory specification, leads to a cascade of errors that halt the entire process. This bug has been observed in various environments, including macOS, Ubuntu, and HPC Linux systems, highlighting its widespread impact. While the exact root cause requires further investigation, potential workarounds include manual lineage selection, checking directory paths, updating software components, and using a consistent environment.

By understanding the specifics of this bug, its impact, and potential solutions, users can better navigate this issue and continue to leverage the power of BUSCO for assessing the completeness of their genomic data. For more information on BUSCO and related tools, please visit BUSCO's official website. This will provide you with further details and updates on the software.