This is a modified pipeline based on the Apogee pipeline (which was modified from the Spaghetti pipeline by Latorre-Pérez et al., 2021) Apogee was authored by Kosal Khun where a new filtering script was modified by Marc-Olivier Duceppe ([email protected]).

My version modifies the minimap2 parameters to include a -f1000 flag (to ensure the minimum fragment length is 1000 bp before mapping) , and modifying the snakefile rules to include "script" instead of "shell" commands when running the fitlerPAF rule. Below is a README on how to use the pipeline and additional details.

ABOUT

The Apogee pipeline is designed for the analysis of Nanopore sequencing data. The pipeline was modified from:

Latorre-Pérez, A., Gimeno-Valero, H., Tanner, K., Pascual, J., Vilanova, C., & Porcar, M. (2021). A Round Trip to the Desert: In situ Nanopore Sequencing Informs Targeted Bioprospecting. Frontiers In Microbiology, 12. doi: 10.3389/fmicb.2021.768240.

The filtering step was replaced to add a filtering option based on mapping quality (mapq).

The pipeline includes modules for quality control, adapter removal, length filtering, chimera detection, taxonomic assignment, and result summarization.

SUMMARY

The Apogee pipeline consists of the following steps:

flowchart TD
    A[Raw .fastq files]
    B{Adapter removal}
    C([Trimmed, .fastq])
    D{Minimum length filtering}
    E([Filtered, .fastq])
    F([Quality check, .txt])
    G{Chimera removal}
    H([Chimera removed, .fastq])
    I{Filter quality}
    J([Filtered, .fastq])
    K{Mapping}
    L([Mapped, .paf])
    M0{Filter paf}
    M1([Filtered, .paf])
    M{Summarize paf}
    N([Otu table, .csv])
    O{Create taxonomic table}
    P([Taxonomy, .csv])

        A-->B-->C-->D-->E & F
        E-->G-->H-->I-->J-->K
        K-->L-->M0-->M1-->M-->N-->O-->P

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USAGE

First, load the conda environment with the required tools:

conda env create -f environment.yaml

Then copy the file config-template.yml as config.yml and customise it to suit your needs.

Finally, to run the pipeline without any parameters:

snakemake --cores <number_of_cores>

NB: You can have multiple config files (one for each set of parameters). To run a specific config file, use:

snakemake --cores <number_of_cores> --configfile <path_to_configfile>

INPUT

You need to provide the pipeline with appropriate 16S sequencing data in FASTQ format.

The pipeline will look for fastq files in the fastq_folder, starting with fastq_prefix (see PARAMETERS):

[FASTQ FOLDER]
    │
    └── {fastq_prefix}_*.fastq

PARAMETERS

The pipeline parameters can be adjusted in the config.yml file. The parameters include:

Parameter	Value
fastq_folder	Path to the folder with FASTQ files
fastq_prefix	Prefix of the fastq files
output_folder	Path to the output folder
mmi	Path to the minimap2 index file
tsv	Path to the taxonomy file
threads	Number of threads to use for parallel processing
min_read_length	Filter on a minimum read length (NanoFilt)
quality_score	Filter on a minimum average read quality score (NanoFilt)
mapping_bp	Stop chain enlongation if there are no minimizers in INT-bp (minimap2)
minibatch_size	Minibatch size for mapping (minimap2)
coverage	If coverage reach this value, region is marked as bad (yacrd)
not_coverage	If the ratio of bad region length on total length is lower than this value, read is marked as NotCovered (yacrd)
filter_paf_block_limit	Hits with a block length alingment lower than this parameter will be removed (filter_paf)
filter_paf_min_confidence	Minimum confidence level (summarize_paf)

OUTPUT

The pipeline generates various output files, including:

[OUTPUT FOLDER]
    │
    ├── porechop
    │       └── {fastq_prefix}_*-porechop.fastq
    ├── nanofilt
    │       └── {fastq_prefix}_*-porechop-nanofilt.fastq
    │       └── {fastq_prefix}_*-porechop-nanofilt.paf (temporary files)
    ├── nanostat
    │       └── {fastq_prefix}_*-porechop-nanofilt-NanoStat.txt
    ├── yacrd
    │       ├── {fastq_prefix}_*-porechop-nanofilt.scrubb.fastq
    │       ├── {fastq_prefix}_*-porechop-nanofilt.scrubb.refilt.fastq
    │       └── {fastq_prefix}_*-porechop-nanofilt.yacrd
    ├── mapping
    │       └── {fastq_prefix}_*-porechop-nanofilt.scrubb.refilt.paf
    ├── filteredPAFs
    │       └── {fastq_prefix}_*-porechop-nanofilt.scrubb.refilt-f.paf
    └── otu_table
            ├── otu_table_{fastq_prefix}.csv
            └── phyloseq_taxonomy_{fastq_prefix}.csv

CREDITS

The original pipeline was written by Adriel Latorre ([email protected]), while the new filtering script was written by Marc-Olivier Duceppe ([email protected]).

CONTRIBUTIONS

Indicate whether the project is open for contributions. If it is, indicate how to contribute.

CITATION

If you use this pipeline for your analysis, please cite it using the CITATION.cff file