Welcome! Here you can find a variety of tools and pipelines tailored to using PacBio HiFi Reads for metagenomics. In addition to the resources currently available, we will continue to add new tools as they are developed.
Several publicly available HiFi shotgun metagenomics datasets are listed on the PacBio-Data page. These include multiple mock communities, reference standards, and empirical samples (human, chicken, and sheep gut microbiome).
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HiFi-MAG-Pipeline: (previously called Genome-Binning-Pipeline) Identify high-quality MAGs from HiFi metagenomic assemblies. Streamlined workflow includes steps with minimap2, MetaBAT2, CheckM, and GTDB-Tk. Outputs high-quality MAG sequences and associated metadata.
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Taxonomic-Functional-Profiling-Protein: Align HiFi reads to a protein database using DIAMOND and prepare inputs for MEGAN-LR, for the purpose of taxonomic and functional profiling. Provides access to taxonomic annotations (NCBI and GTDB), and allows functional annotations based on several databases (EC, SEED, InterPro2GO, eggNOG). Easily summarize taxonomic and functional results using MEGAN-RMA-Summary.
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Taxonomic-Profiling-Nucleotide: Align HiFi reads to a nucleotide database using minimap2 and prepare inputs for MEGAN-LR, for the purpose of taxonomic profiling. This only provides access to NCBI taxonomic annotations. Easily summarize taxonomic results using MEGAN-RMA-Summary. If you are attempting to identify microbial contamination reads in targeted sequencing datasets, use this pipeline!
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MEGAN-RMA-Summary: Obtain functional and taxonomic class count summaries for a set of RMA files created from the above MEGAN-LR pipelines. Outputs absolute read counts and normalized read counts across samples for classes of each database, and summarizes the proportion of HiFi reads receiving functional and taxonomic annotations. New feature: Outputs NCBI taxonomic counts in kraken (kreport) and metaphlan (mpa) formats. Workflows are available for both protein-based RMA files and for nucleotide-based RMA files.
Each of these pipelines can be found in their respective folders. They are made available as Snakemake workflows. Snakemake is a python-based workflow manager. Snakemake workflows are highly portable because dependencies and environments are automatically setup using Anaconda/Conda. Snakemake also allows reproducibility, checkpointing, and the ability to scale workflows using HPC and cloud environments. Snakemake v5+ is required, and the workflows have been tested using v5.26+.
Scripts and tools for metagenomics tasks are available in the pb-metagenomics-scripts folder. Currently, there are several scripts to convert the outputs from metagenomics taxonomic profiling programs into standard formats, including kraken report (kreport) and metaphlan report (mpa) formats. There is also a Jupyter notebook that demonstrates how to analyze and compare kraken report files for multiple samples.
All documentation for snakemake pipelines can be found in the docs/ folder above. Links are also provided below.
Available pipeline documentation:
- Tutorial: HiFi-MAG-Pipeline
- Tutorial: Taxonomic-Functional-Profiling-Protein
- Tutorial: Taxonomic-Profiling-Nucleotide
- Tutorial: MEGAN-RMA-Summary
The documentation for pb-metagenomics-scripts is provided in the folder.
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