created 27.01.2020 last edit: 21.06.2020 by Marc Ruebsam

Project Folder: 16S_Metabarcoding Description: Microbiota profiling with long amplicons using nanopore sequencing: This folder is used for 16S Metabarcoding analysis using the Nanopore MinION platform. It contains subfolders for storage of raw data (created during the sequencing run), intermediate processing steps, analysis results and reports (created by the pipeline). The first three are organized per run (unique run ID), the last one is separated by time points and sample IDs. Additional information regarding the origin and processing of each sample can be received from the METADATA directory. The pipeline used for analysis of the data is based on the snakemake files in the Pipeline directory. Documentation contains written evaluation of the results, presentations and references. Type of Data: Fast5/Fastq/Pictures/Report files Participants: Silke Grauling-Halama (silke.grauling-halama@nct-heidelberg.de), Pornpimol Charoentong (Pornpimol.Charoentong@nct-heidelberg.de), Marc Ruebsam (marc.ruebsam@bioquant.uni-heidelberg.de) Correspondence: Silke Grauling-Halama (silke.grauling-halama@nct-heidelberg.de)

The raw data is created by the MinKNOW software during the MinION run. Crucial information regarding the samples, library preparation and sequencing run are stored inside METADATA/EXPERIMENT_SEQUENCING.xlsx file.

== Sequencing with MinKNOW ==

• Follow the protocol for flow cell preparation and loading of the library
  • Note down the number of pores during the flow cell check
• Select the folder specified as Project (see above) as output directory (will always be a "00_raw_data" folder inside the project specific folder)
• Specify a experiment and sample name (as above)
  • Please do not use spaces or special characters (e.g. äöüß.,<>|), except "_"
• Activate VBZ compression
• Deactivate live basecalling
• Start the sequencing run
• MinKNOW will create the output directory inside 00_raw_data according to the experiment and sample names specified
  • if you want to create and save PDF reports, create a folder inside the Run ID folder called "reports" (the same folder that contains the fast5 folder)
  • move PDFs in there

== After the run has finished ==

• Make sure the sequencing run has finished successfully and the raw data is in the expected location
• Close all open folders and files related to the current run

MeBaPiNa (metabarcoding analysis pipeline for Nanopore datasets) is a pipeline implemented in snakemake. It takes raw fast5 files and automatically processes it according to the specifications in the METADATA/PIPELINE_CONFIG.yaml file. Statistics and figures for the requested samples are reported in the 03_report directory.

== Installing snakemake ==

• Has to be done once on the machine used for running the Pipeline
  • PLEASE THINK BEFORE COPY & PASTING
  • Install conda

    update machine

    sudo yum update -y sudo yum upgrade -y sudo yum install -y epel-release ## EPEL repository for yum sudo yum install -y cifs-utils ## mounting of windows network shares sudo yum install -y wget ## querry downloads sudo yum install -y git ## git

    get lateste miniconda

    wget https://repo.continuum.io/miniconda/Miniconda3-latest-Linux-x86_64.sh -O miniconda.sh

    create directory for miniconda owed by user

    sudo mkdir -p /opt/miniconda pid_me=$(id --user) gid_me=$(id --group) sudo chown ${pid_me}:${gid_me} /opt/miniconda unset pid_me gid_me

    execute installation script

    bash miniconda.sh -b -p /opt/miniconda/miniconda3 export PATH="/opt/miniconda/miniconda3/bin:$PATH" rm miniconda.sh

    update and initialize

    hash -r conda update -y -q conda conda init bash
  • Install snakemake (create snakemake environment with conda)

    create conda environment of snakemake

    conda create -y -c defaults -c bioconda -c conda-forge -n snakemake snakemake xlrd ## alternatively use the configuration for 5.10 conda env create -f Pipeline/MeBaPiNa/envs/snakemake.yml -n snakemake

    remove temp files

    conda clean -y --all
    • Install guppy standalone - download guppy from https://europe.oxfordnanoportal.com/software/analysis/ont_guppy_3.4.1-1~xenial_amd64.deb - install

== Before running the Pipeline ==

• Update the METADATA/PIPELINE_CONFIG.yaml file
  • Experiment specifications
    • project, tmp: build the path to the project directory like {project}/{tmp}
    • meta: path to the EXPERIMENT_SEQUENCING.xlsx inside the project directory like {project}/{tmp}/{meta}
    • log: path to the ANALYSIS_PROGRESS_MANAGEMENT.csv inside the project directory like {project}/{tmp}/{log}
    • samples: list of sample names from the EXPERIMENT_SEQUENCING.xlsx to analze
  • Methods for analysis
    • methodologie: list of method abbreviations to use for analysis
  • Workstation specifications
    • gpu: whether or not a guppy compatible GPU is available
  • Filtering options
    • q_min: minimal quality score for read filtering
    • len_min: minimal length for read filtering
    • len_max: maximal length for read filtering
    • min_featurereads minimum number of reads per cluster or taxon
    • min_readidentity minimum read identity for clustering (OTU)
    • min_confidence confidence used for classification of taxa at {refrank} level
    • plot_sample down-sampling reads in plots to this number to reduce computational complexity (distorts statistics shown in some plots)
  • Reference database (currently only {refsource} is implemented)
    • source: which reference database to use
    • rank: at which rank the taxonomy should be determined

== Run MeBaPiNa ==

I. Run full pipeline

• Activate environment if used conda is used for snakemake ## activate conda environment conda activate snakemake
• Change directory into the project directory (or use absolute paths below)
• DRY RUN: Optionally execute the pipeline as a dry run showing all rules that are going to be executed
  • the '-n' invokes a dry run and '-pr' includes higher verbosity
  • change the path to the config file and Pipeline snakefile if necessary

    dry run pipeline with verbosity

    snakemake -npr --use-conda --cores 'all' --configfile METADATA/PIPELINE_CONFIG.yaml --snakefile Pipeline/MeBaPiNa/Snakefile
• Execute the pipeline
  • change the path to the config file and Pipeline snakefile if necessary

    run pipeline

    snakemake -pr --use-conda --cores 'all' --configfile METADATA/PIPELINE_CONFIG.yaml --snakefile Pipeline/MeBaPiNa/Snakefile
• Please note that the --cores 'all' might not work to request all available threads at the machine and has to be changed to an integer manually

II. Perform only basecalling

• In case you only want to basecall the data and have a look at the data before running the full pipeline
• Or in case you want to perform basecalling on a separate machine than the rest of the pipeline
  • you can run

    run the pipeline only until basecalling has been finished

    snakemake -pr --use-conda --cores 'all' --configfile METADATA/PIPELINE_CONFIG.yaml --snakefile Pipeline/MeBaPiNa/Snakefile only_basecall
  • output will be generated inside the 03_report/ dorectory and ccan be reviewed
  • please note that this instance of the pipeline has to finish before you can start further analysis by invoking the command from I.
  • in other words: no dependecy between this instance and another can be established automatically and has to be insured manually

III. Update report file / add missing reports

• In case you want to update the log file ANALYSIS_PROGRESS_MANAGEMENT.csv
  • you can run

    rerun report rules and any of its upwards dependencies

    snakemake -pr --use-conda --cores 'all' --configfile METADATA/PIPELINE_CONFIG.yaml --snakefile Pipeline/MeBaPiNa/Snakefile update_report

IV. Rerun certain step

• You can rerun a certain step in the pipeline or reproduce a certain file (e.g. if it was corrupted)
  • find the rule corresponding to the processing step in question
    • check the output location for the output files
    • if the output files still exist, delete or move them (rerunning only works if the file doesn't already exist)
  • get the path to the desired output file and call snakemake with it, e.g.:

    rerun krona plot for one of the samples

    snakemake -pr --use-conda --cores 'all' --configfile METADATA/PIPELINE_CONFIG.yaml --snakefile Pipeline/MeBaPiNa/Snakefile 16S_Metabarcoding/02_analysis_results/03_kmer_mapping/20191007_1559_MN31344_FAK76605_2bf006ff/barcode06/silva_Species/krona.html
  • you can list multiple files to rerun all of them
• Note! rerunning of an intermediate step does not automatically update all below dependencies
  • if this is desired, proceed as above, by deleting or moving the file in question
  • then call the pipeline as normal

== After running the pipeline ==

• Check the snakemake log file (in .snakemake/logs/) for any errors
• Repeat a dry run to check that only the "all_target" rule is left (this rule will be executed every time)
• check the results inside the 03_report directory

The following list gives an overview of the files inside the 16S_Metabarcoding directory. The list does not include temporary files or log files. Please have a look at the Documentation/ThesisManuscript/Figures/DAG.pdf for an overview of the rule dependencies.

== METADATA ==

./METADATA/

• directory containing important metadata files and reference sequences

  PIPELINE_CONFIG.yaml

  • configurations of the pipeline
  • see above

  Reference_Sequences/

  • directory containing reference sequences

    primers/

    • primers used for the extraction of the amplicon region from the references

    {refsource}/

    • directory containing the reference database files and subdirectories for the methodology specific files

      rule download_reffiles and construct_refseq:

      reference.fasta

      • reference sequences trimmed to amplicon region, filtered by length and removed duplicates (100% identity)

      rule indexing_reference:

      reference.mmi

      • reference file for alignments with minimap2

        rules construct_reftax and building_database:

        kraken2/

        • kraken2 and bracken specific reference database files

        rules construct_reftax, q2import_reftax, q2import_refseq and q2train_classifyer:

        qiime/

        • qiime specific reference database files
        • these files might be incomplete due to an unsuccessful construction

        rules construct_reftax:

        krona/

        • krona specific reference database files

    lambda/

    • lambda phage reference sequence

    zymobiomics/

    • reference files only containing only species present in the zymobiomics mock community

== Pipeline ==

./Pipeline/

• a directory containing software and scripts associated with the pipeline

  MeBaPiNa/

  • directory containing files required to run the pipeline

    Snakefile

    • central file of the pipeline
    • starts execution and sources other files

    envs/

    • conda environment files used in the pipeline

    rules/

    • definition of rules used in the pipeline

    scripts/

    • scripts used in the rules
    • some additional scripts

== Raw data ==

./00_raw_data/

• directory containing raw sequencing files of all runs

• priority:

  • these files are of highest priority and cannot be replaced

  rule move_raw:

  {run}/

  • run specific sub-directory

    fast5/

    • directory containing raw fast5 sequencing files

    reports/

    • optional directory with run reports as PDFs
    • created manually in MinKNOW (feature automation was promised by ONT)

== Intermediate processing files ==

./01_processed_data/

• directory containing intermediate processing files of all steps and runs

• priority:

  • these files are produced during key steps in the analysis processes
  • they can be replaced if the files from ./00_raw_data/ are still present, but this might require a considerable amount of recomputation
  • they are required in case some steps should be reran (e.g. with new parameters)

  01_basecalling/

  • directory containing the basecalled raw-reads of all runs

    {run}/

    • run specific sub-directory

      rule basecall_raw:

      guppy_basecaller_logs/

      • logs from basecaller sequencing_summary.txt
      • table with information about all basecalled reads pass/
      • directory containing basecalled fastq files of all barcodes {barcode}
        • sub-directories with fastq files assigned to the barcode unclassified
        • sub-directory with fastq files without detected barcode (contains all files if library wasn't multiplexed)

      rule sorting_seqsum_barc and splitting_seqsum_barc

      sequencing_summary/

      • same information as in sequencing_summary.txt sequencing_summary_sorted.txt
        • but sorted by barcode column and start time split/
        • and as separate files per barcode (can be used to extract raw reads per barcode)

  02_trimming_filtering/

  • directory containing the trimmed and filtered basecalled-reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        rule trim_basecalled

        trimmed.fastq

        • trimming and second demultiplexing of basecalled reads other/
        • reads assigned to other barcodes during second demultiplexing
        • these reads are not used for anything and could be deleted

        rule filter_trimmed:

        filtered.fastq

        • length and quality filtered filtered reads

  03_alignment/

  • directory containing the alignment of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}/

        • reference database specific sub-directory

          rule aligning_filtered and filter_aligned:

          filteredsorted.bam

          • filtered alignement of the trimmed and filtered reads filteredsorted.bam.bai
          • index of bam

  03_kmer_mapping/

  • directory containing the k-mer mapping of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}_{refrank}/

        • reference database and rank specific sub-directory

          rule kmermap_filtered:

          filtered.kraken2

          • per read classification information filtered.kreport2
          • per taxa classification information

          rule retax_kmermap:

          Species.bracken

          • per taxa reestimation information Species.kreport2
          • per taxa classification information after reestimation

  03_otu_picking/

  • directory containing the clustered OTUs and the taxonomic assignmnts of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}/

        • reference database specific sub-directory

          rule generate_manifestfile, q2import_filtered, q2derep_imported, q2otupick

          cluster_centseq.qza

          • center sequence per OTU of the trimmed and filtered reads cluster_ftable.qza
          • table with counts per OTU of the trimmed and filtered reads cluster_newrefseq.qza
          • new reference sequences
          • can be used for subsequent runs of open-reference clustering for consistent definitions of features across open-reference feature tables.

          q2uchime_otus and q2filter_uchime:

          filt_ftable.qza

          • table with counts per OTU after chimera removal and min coverage filtering filt_centseq.qza
          • center sequence per OTU after chimera removal and min coverage filtering

          {other directories}

          • storage location for temporary files during conversion

        {refsource}_{refrank}/

        • reference database and rank specific sub-directory

          rule rereplicate_q2filter and kmermap_q2rereplicate:

          filtered.kraken2

          • per OTU classification information filtered.kreport2
          • per taxa classification information

== Result files ==

./02_analysis_results/

• directory containing result files of all steps and runs

• priority:

  • these files are of low priority
  • they can easily be replaced if the files from ./01_processed_data/ are still present
  • important files are copied to ./03_report/

  01_basecalling/

  • directory containing plots and statistics of the basecalled raw-reads of all runs

    {run}/

    • run specific sub-directory

      rule plot_fastq_pipe_basecall:

      fastqc/

      • read QC: all passed reads

      rule plot_nanocomp_seqsum_basecall:

      nanocomp/

      • barcode QC: per barcode

      rule plot_nanoplot_seqsum_basecall:

      nanoplot/

      • general QC: all reads, including calibtation strads

      rule plot_nanoqc_fastq_basecall:

      nanoqc/

      • per base QC: all reads

      rule plot_pycoqc_seqsum_basecall:

      pycoqc/

      • general QC: all reads

  02_trimming_filtering/

  • directory containing plots and statistics of the trimmed and filtered basecalled-reads of all runs

    {run}/

    • run specific sub-directory

      rule plot_fastqc_fastq_filter:

      fastqc/

      • read QC: trimmed and filtered barcoded reads

      rule plot_nanocomp_fastq_filter:

      nanocomp/

      • barcode QC: trimmed and filtered barcoded reads

      rule plot_nanoplot_fastq_filter:

      nanoplot/

      • general QC: trimmed and filtered barcoded reads

      rule plot_fastq_pipe_filter:

      nanoqc/

      • per base QC: trimmed and filtered barcoded reads

  03_alignment/

  • directory containing plots and statistics of the alignment of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}/

        • reference database specific sub-directory

          rule plot_refseq_coverage:

          covdist.pdf covpos.pdf - coverage of reference sequences - covdist.pdf seem broken when executing on the VM

          rule plot_pycoqc_aligned:

          pycoqc.html pycoqc.json - alignment QC

        {refsource}_{refrank}/

        • reference database and rank specific sub-directory

          rule counttax_aligned:

          aligned.counttaxlist - taxonomic classification

          rule plot_krona_aligned_text:

          krona.html - visualization of taxonomic classification

  03_kmer_mapping/

  • directory containing plots and statistics of the k-mer mapping of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}_{refrank}/

        • reference database and rank specific sub-directory

          rule counttax_kmermap:

          kmer.counttaxlist - taxonomic classification

          rule plot_krona_kmermap_kraken:

          krona.html - visualization of taxonomic classification

          rule plot_krona_kmermap_text:

          krona_bracken.html - visualization of taxonomic classification after reestimation

  03_otu_picking/

  • directory containing plots and statistics of the clustered OTUs and the taxonomic assignmnts of trimmed and filtered reads of all runs

    {run}/

    • run specific sub-directory

      {barcode}/

      • barcode specific sub-directory

        {refsource}/

        • reference database specific sub-directory

          rule plot_qiime2_q2otupick:

          q2otupick
          - clustered reads overview

          rule plot_qiime2_q2filter:

          q2filter
          - clustered reads overview after filtering

        {refsource}_{refrank}/

        • reference database and rank specific sub-directory

          rule counttax_q2kmermap:

          kmer.counttaxlist - taxonomic classification

          rule plot_krona_q2rerep:

          krona.html - visualization of taxonomic classification

== Report per sample ==

./03_report/

• directory containing result files, plots and statistics per timepoint and sample

• priority:

  • the files in this directory are of high priority
  • they can be recreated if the files from ./01_processed_data/ and ./02_analysis_results/ are still presentat
  • they represent the final output of the pipeline

  Reference_Sequences/

  • directory with plots and statistics of reference database

    {refsource}/

    • reference database specific sub-directory

      rule stat_refseq_lenstat:

      reference_lengthdist.pdf reference_lengthdist.tsv

      • length of reference sequences

      rule stat_refseq_taxaranks

      reference_taxaranks.tsv

      • distribution of reference taxa ranks

  {timepoint}/

  • timepoint specific folder or non-PROMISE

    {sample}/

    • sample specific folder

      {run}-{barcode}/

      • sample per run specific folder

      • in case the same sample is sequenced in multiple runs

        rule stat_general_readbasecount:

        read_base_counts.tsv

        • statistics of reads and bases of raw, basecalled, trimmed and filtered reads

        rule :

        03_alignment-{refsource}-alignment_rates.tsv

        • aligment statistics and error rates

        rule stat_otu_feature:

        03_otu_picking-{refsource}-feature_counts.tsv

        • number of clusters and reads before and after filtering

        rule stat_align_taxa:

        03_alignment-{refsource}_{refrank}-taxa_counts.tsv

        • statistics of classified taxa and reads

        rule stat_kmer_taxa:

        03_kmer_mapping-{refsource}_{refrank}-taxa_counts.tsv

        • statistics of classified taxa and reads

        rule stat_kmer_retaxa:

        03_kmer_mapping-{refsource}_{refrank}-retaxa_counts.tsv

        • statistics of classified taxa and reads after reestimation

        rule stat_otu_taxa:

        03_otu_picking-{refsource}_{refrank}-taxa_counts.tsv

        • statistics of classified taxa and reads

        rule stat_align_taxa_diversity:

        03_alignment-{refsource}_{refrank}-taxa_covdist.pdf

        • distribution of taxa abundance 03_alignment-{refsource}_{refrank}-taxa_diversity.tsv
        • diversity, richness and evenness of community

        rule stat_kmer_taxa_diversity:

        03_kmer_mapping-{refsource}_{refrank}-taxa_covdist.pdf

        • distribution of taxa abundance 03_kmer_mapping-{refsource}_{refrank}-taxa_diversity.tsv
        • diversity, richness and evenness of community

        rule stat_otu_taxa_diversity:

        03_otu_picking-{refsource}_{refrank}-taxa_diversity.tsv

        • distribution of taxa abundance 03_otu_picking-{refsource}_{refrank}-taxa_covdist.pdf
        • diversity, richness and evenness of community

        {other files}

        • are copies from the similarly named files in the ./02_analysis_results/ directory