cjneely10/EukMetaSanity

Structural and functional annotation of eukaryotic metagenome-assembled genomes

EukMetaSanity

About

Eukaryotic genome annotation is a laborious and time-intensive process. EukMetaSanity provides a structural and functional annotation of MAGs in a highly-parallel fashion, allowing for quick and in-depth analyses.

This software suite is broken up into several sub-programs

Run

Perform high-quality structural annotation using protein evidence recruited from the Orthologous Database of Proteins and the Marine Microbial Eukaryotic Transcriptomic Sequencing Project databases.

Performs:

• Taxonomy prediction using proteins identified by MetaEuk
• DNA repeat modeling and masking using RepeatModeler and RepeatMasker, respectively
• Ab initio prediction with Augustus and/or GeneMark

Refine

Map RNA-seq (using HISAT2) and assembled transcriptome (using GMAP) evidence from closely related organisms (same organism or species) to the genome to add additional evidence using BRAKER2.

Report

Search KEGG, EggNOG, and any MMseqs2 database for functional annotation of putative proteins.

Check the quality of your annotation using BUSCO.

Installation

See INSTALLATION.md for detailed installation instructions.

Using yapim configuration files

EukMetaSanity is built using the YAPIM library, which operates through a configuration file that is provided with each pipeline.

Running a YAPIM pipeline typically consists of copying a default configuration file to your working directory, making edits to fit your resource and analysis needs, and running the pipeline on a folder of input files.

At the top of each configuration file will be a section that defines total available resources:

###########################################
## Pipeline input section
INPUT:
  root: all

## Global settings
GLOBAL:
  # Maximum threads/cpus to use in analysis
  MaxThreads: 20
  # Maximum memory to use (in GB)
  MaxMemory: 120

###########################################

Provide the maximum threads and memory to allot towards the analysis.

Within each section of the configuration file, set the resources to allot to each input genome

Taxonomy:
  # Number of threads task will use
  threads: 5
  # Amount of memory task will use (in GB)
  memory: 10
  time: "8:00:00"

For example, based on the provided maximum resource limits, the preceding section can run up to 4 genomes at a time.

When launched using SLURM, the maximum resources can be set quite high, and the task-level resources can be set to match node resource limits that exist on your systems.

---

We have provided a test set of data for use in validating installation, or as a means of better understanding the EukMetaSanity implementation.

These files are present in the directory tests/data:

tests/
  |-- data/
    |-- NAO-all-DCM-20-180-00_bin-1.fna
    |-- NAO-all-DCM-20-180-00_bin-2.fna
    |-- NAO-all-DCM-20-180-00_bin-19.fna

If you are using your own input set, ensure that your FASTA files' extensions are one of the following:

.fna
.fa
.fasta
.faa
.fas

Run

Config setup

Copy and edit the run-config.yaml config file to fit your analysis needs.

cp $EukMS_run/run-config.yaml ./

In the GLOBAL section, you will want to set the number of MaxThreads you will use to run the analysis, as well as the MaxMemory to be used. If using SLURM, set this to be appropriately high (> 100 threads, etc.).

In the SLURM section, set USE_CLUSTER to true if needed, and provide run configuration details (such as qos, job_name, partition, account, etc.). Make sure the id listed in user-id matches your linux id.

In each subsequent section, you may adjust the threads, memory, and FLAGS that are passed to the program. Be sure to set the time allocation for each step if running pipeline on SLURM.

If you choose to omit running AbinitioAugustus or AbinitioGeneMark, set its respective skip flag to be true.

---  # document start

###########################################
## Pipeline input section
INPUT:
  root: all

## Global settings
GLOBAL:
  # Maximum threads/cpus to use in analysis
  MaxThreads: 20
  # Maximum memory to use (in GB)
  MaxMemory: 120

###########################################

SLURM:
  ## Set to true if using SLURM
  USE_CLUSTER: false
  ## Pass any flags you wish below
  ## DO NOT PASS the following:
  ## --nodes, --ntasks, --mem, --cpus-per-task
  --qos: unlim
  --job-name: EukMS
  user-id: uid

MetaEukEV:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 100
  time: "12:00:00"
  dependencies:
    MetaEuk:
      program: metaeuk
      data:
        /path/to/odb-mmetsp_db
      # Pass any flags to metaeuk required
      FLAGS:
        --min-length 30
        --metaeuk-eval 0.0001
        -s 5
        --cov-mode 0
        -c 0.3
        -e 100
        --max-overlap 0
        --remove-tmp-files

Taxonomy:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 100
  time: "8:00:00"
  dependencies:
    MMSeqsCreateDB:
      program: mmseqs
      threads: 1

    MMSeqsTaxonomy:
      program: mmseqs
      cutoff: 8.0  # Minimum % of mapped reads to tax level
      data:
        /path/to/odb-mmetsp_db
      # Pass any flags to mmseqs required
      FLAGS:
        --remove-tmp-files
        -s 6.5
        --min-seq-id 0.40
        -c 0.3
        --cov-mode 0

Repeats:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 16
  time: "24:00:00"
  dependencies:
    RModBuildDatabase:
      time: "10:00"
      threads: 1
      program: BuildDatabase

    RModRepeatModeler:
      program: RepeatModeler
      skip: false

    RMaskRepeatMasker:
      program: RepeatMasker
      level: family
      data:
        "" # Comma-separated list of repeat models to incorporate
      FLAGS:
        -nolow

    RMaskProcessRepeats:
      time: "30:00"
      threads: 1
      program: ProcessRepeats
      FLAGS:
        -nolow

    RMaskRMOut:
      time: "10:00"
      threads: 1
      program: rmOutToGFF3.pl

AbinitioGeneMark:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 16
  time: "16:00:00"
  skip: false
  dependencies:
    MMSeqsFilterTaxSeqDB:
      threads: 1
      time: "10:00"
      program: mmseqs
      level: order
      data:
        /path/to/odb-mmetsp_db

    GeneMarkProtHint:
      program: prothint.py

    GeneMarkPETAP:
      program: gmes_petap.pl
      FLAGS:
        --min_contig 100
        --max_contig 1000000000
        --max_gap 5000
        --max_mask 5000
        --min_contig_in_predict 100
        --min_gene_in_predict 10
        --gc_donor 0.001
        --max_intron 10000
        --max_intergenic 50000
        --soft_mask auto

AbinitioAugustus:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 8
  time: "24:00:00"
  skip: false
  dependencies:
    Augustus:
      program: augustus
      cutoff: 25.0
      rounds: 2

Tier:
  # Number of threads task will use
  threads: 1
  # Amount of memory task will use (in GB)
  memory: 2
  time: "10:00"
  program: locus_solver
  tier: 1

...  # document end

Call pipeline

---

Note on HPC usage: EukMetaSanity launches multiple SLURM jobs to complete its analysis, similar to SLURM job arrays. The node on which EukMetaSanity is launched manages these jobs - RAM and CPU usage is minimal. Users should follow their institution's best practices for selecting a node on which to launch this program.

---

Activate your EukMS_run conda environment.

conda activate EukMS_run

Ensure your input FASTA sequences do not have the pipe (|) character present.

Run the pipeline using the command:

yapim run -i /path/to/EukMetaSanity/tests/data -c run-config.yaml -p $EukMS_run

If run with default config parameters, the analysis should complete within about 4 hours. This can be sped up by running the analysis on SLURM and/or increasing the thread count.

Run output

This will create a directory structure with non-empty files resembling:

out/
  |-- wdir/
  |-- input/
  |-- run-eukmetasanity.log
  |-- results/
    |-- run/
        |-- run.pkl
        |-- NAO-all-DCM-20-180-00_bin-1
          |-- NAO-all-DCM-20-180-00_bin-1.n.Tier.gff3  # Tier n final results (GFF3)
          |-- NAO-all-DCM-20-180-00_bin-1.n.Tier.faa  # Tier n final results (FASTA)
          |-- NAO-all-DCM-20-180-00_bin-1.AbinitioAugustus.gff3  # Augustus results (GFF3)
          |-- NAO-all-DCM-20-180-00_bin-1.AbinitioAugustus.faa  # Augustus results (FASTA)
          |-- NAO-all-DCM-20-180-00_bin-1.AbinitioGeneMark.gff3  # GeneMark results (GFF3)
          |-- NAO-all-DCM-20-180-00_bin-1.AbinitioGeneMark.faa  # GeneMark results (FASTA)
          |-- NAO-all-DCM-20-180-00_bin-1.MetaEukEV.gff3  # MetaEuk results (GFF3)
          |-- NAO-all-DCM-20-180-00_bin-1.MetaEukEV.faa  # MetaEuk results (FASTA)
          |-- NAO-all-DCM-20-180-00_bin-1.Repeats.gff3  # Repeats locations (GFF3)
          |-- NAO-all-DCM-20-180-00_bin-1.Repeats.tbl  # Summary of repeats annotation
          |-- NAO-all-DCM-20-180-00_bin-1.Repeats.fna  # Masked input genome (FASTA)
          |-- NAO-all-DCM-20-180-00_bin-1.Taxonomy.txt  # Taxonomy assignment summary
        |-- NAO-all-DCM-20-180-00_bin-19/
          ...
        |-- NAO-all-DCM-20-180-00_bin-2/
          ...

Installation check:

To confirm proper GeneMark installation, users should run the following command:

ls out/wdir/*/AbinitioGeneMark.GeneMarkPETAP/*.sh

You should see the following files:

out/wdir/NAO-all-DCM-20-180-00_bin-19/AbinitioGeneMark.GeneMarkPETAP/gmep.sh
out/wdir/NAO-all-DCM-20-180-00_bin-19/AbinitioGeneMark.GeneMarkPETAP/gmes.sh
out/wdir/NAO-all-DCM-20-180-00_bin-1/AbinitioGeneMark.GeneMarkPETAP/gmep.sh
out/wdir/NAO-all-DCM-20-180-00_bin-2/AbinitioGeneMark.GeneMarkPETAP/gmep.sh

If you do not see files named gmep.sh, there may be an issue with your ProtHint installation. Check your PATH to confirm that GeneMark-related directories are present, and check the version of diamond that is present in the gmes_linux64/ProtHint/dependencies and confirm that it runs as expected.

Note on running:

EukMetaSanity will not re-run already completed steps within a given pipeline. If you would like to re-do a particular portion of the pipeline, simply delete its directories in the project structure. For example, to redo the Taxonomy step of the run pipeline for all MAGs, run the following command to delete all existing data:

yapim clean -p $EukMS_run Taxonomy -o out

The preceding command will not only delete the results generated by the Taxonomy step, but will also remove all other steps in the pipeline that use these results. If you only wish to delete a step and nothing else, run:

rm -r out/wdir/*/Taxonomy*

---

Refine (optional)

For the Refine pipeline, we do not provide transcriptomic data for testing. If you wish to test this installation, you must provide your own set of test data.

Config setup

Copy and edit the refine-config.yaml config file to fit your analysis needs.

cp $EukMS_refine/refine-config.yaml ./

As before, set resource usage in the GLOBAL settings as well as for each subsequent section. Also set the SLURM settings, if needed.

Pay close attention to the input format for RNA-seq and transcriptomes that is required by the config file:

# Paths to RNA-seq should be contained in a file with the format (excluding spaces around tab):
genome-file-basename \t /path/to/r1.fq,/path/to/r2.fq;/path/to/r3.fq,/path/to/r4.fq
# Transcriptomes should be contained in a file with the format (excluding spaces around tab):
genome-file-basename \t /path/to/tr1.fna,/path/to/tr2.fna

The listed paired-end or single-end reads will be mapped to the file that begins with file-basename, as will the list of transcriptomes.

---  # document start

###########################################
## Pipeline input section
INPUT:
  run: all

## Global settings
GLOBAL:
  # Maximum threads/cpus to use in analysis
  MaxThreads: 24
  # Maximum memory to use (in GB)
  MaxMemory: 100

###########################################

SLURM:
  ## Set to true if using SLURM
  USE_CLUSTER: false
  ## Pass any flags you wish below
  ## DO NOT PASS the following:
  ## --nodes, --ntasks, --mem, --cpus-per-task
  --qos: unlim
  --job-name: EukMS
  user-id: uid

CollectInput:
  # Number of threads task will use
  threads: 1
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  # Should be in format (excluding spaces around tab):
  # genome-file-basename \t /path/To/tr1.fna[,/path/To/tr2.fna]
  transcriptomes: /path/To/transcriptome-mapping-file
  # Should be in format (excluding spaces around tab):
  # genome-file-basename \t /path/To/r1.fq[,/path/To/r2.fq][;/path/To/r3.fq[,/path/To/r4.fq]]
  rnaseq: /path/To/rnaseq-mapping-file

GatherProteins:
  # Number of threads task will use
  threads: 8
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  dependencies:
    MMSeqsFilterTaxSeqDB:
      program: mmseqs
      level: order
      data:
        /path/to/odb-mmetsp_db

Transcriptomes:
  # Number of threads task will use
  threads: 8
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  dependencies:
    GMAPBuild:
      threads: 1
      program: gmap_build

    GMAP:
      program: gmap
      FLAGS:
        -B 5
        --input-buffer-size 1000000
        --output-buffer-size 1000000
        -f samse

RNASeq:
  # Number of threads task will use
  threads: 8
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  dependencies:
    Hisat2Build:
      threads: 1
      program: hisat2-build

    Hisat2:
      program: hisat2
      FLAGS:
        ""

MergeSams:
  # Number of threads task will use
  threads: 1
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"

ProcessMapping:
  # Number of threads task will use
  threads: 8
  # Amount of memory task will use (in GB)
  memory: 60
  time: "4:00:00"
  dependencies:
    SambambaSort:
      program: sambamba

RunBraker:
  # Number of threads task will use
  threads: 4
  # Amount of memory task will use (in GB)
  memory: 100
  time: "4:00:00"
  dependencies:
    Braker:
      program: braker.pl
      FLAGS:
        # Provide flags as desired
        # Currently, `exonerate` is the only supported protein mapper
        "--prg=exonerate"

...  # document end

Call pipeline

Activate your EukMS_refine conda environment.

conda activate EukMS_refine

Run pipeline with the command:

yapim run -i /path/to/EukMetaSanity/tests/data -c refine-config.yaml -p $EukMS_refine

Refine output

This will update the directory structure:

out/
  |-- input/
  |-- wdir/
  |-- refine-eukmetasanity.log
  |-- run-eukmetasanity.log
  |-- results/
      |-- refine/
          |-- refine.pkl
          |-- mag1/
              |-- mag1.nr.gff3  # Final predictions
              |-- mag1.cds.fna  # CDS sequences
              |-- mag1.faa  # Protein sequences
              |-- augustus.hints.gtf  # Augustus predictions
              |-- genemark.gtf  # GeneMark predictions
          |-- mag2/
              ...
      |-- run/
          ...

Report (optional)

Config setup

Copy and edit the report-config.yaml config file to fit your analysis needs.

cp $EukMS_report/report-config.yaml ./

As before, set resource usage in the GLOBAL settings as well as for each subsequent section. Also set the SLURM settings, if needed.

Set skip to true if you wish to skip any of the steps listed in the pipeline. If running EggNog, ensure to provide the path to the eggnog databases on your system.

If you wish to use the results of the refine pipeline instead of default results from the run pipeline, update the INPUT section as follows:

INPUT:
  root: all
  refine: all

Otherwise, set the protein output you wish to annotate:

---  # document start

###########################################
## Pipeline input section
INPUT:
  root: all
  run:
    prot: merged-prot  # or genemark-prot or aug-prot or evidence-prot
#  refine:  # Uncomment these two lines, and comment out the two preceding lines,
#    prot: prot  #  to annotate results from `refine` pipeline

## Global settings
GLOBAL:
  # Maximum threads/cpus to use in analysis
  MaxThreads: 20
  # Maximum memory to use (in GB)
  MaxMemory: 100

###########################################

SLURM:
  ## Set to true if using SLURM
  USE_CLUSTER: false
  ## Pass any flags you wish below
  ## DO NOT PASS the following:
  ## --nodes, --ntasks, --mem, --cpus-per-task
  --qos: unlim
  --job-name: EukMS
  user-id: uid

Quality:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  skip: false
  dependencies:
    Busco:
      program: busco
      mode: prot
      lineage: eukaryota
      FLAGS:
        -f

RRNASearch:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 90
  time: "4:00:00"
  skip: false
  dependencies:
    MMSeqsCreateDB:
      program: mmseqs

    MMSeqsSearch:
      data:
        /path/to/SILVA
      program: mmseqs
      subname: search
      FLAGS:
        -c 0.3
        --cov-mode 1
        --remove-tmp-files
        --search-type 3

    MMSeqsConvertAlis:
      data:
        /path/to/SILVA
      program: mmseqs

ProteinAnnotation:
  # Number of threads task will use
  threads: 16
  # Amount of memory task will use (in GB)
  memory: 90
  time: "4:00:00"
  skip: false
  dependencies:
    MMSeqsCreateDB:
      program: mmseqs

    MMSeqsSearch:
      data:
        /path/to/odb-mmetsp_db
        p:/path/to/UniProtKB_Swiss-Prot
      program: mmseqs
      subname: linsearch
      FLAGS:
        -c 0.3
        --cov-mode 1
        --remove-tmp-files

    MMSeqsConvertAlis:
      data:
        /path/to/odb-mmetsp_db
        p:/path/to/UniProtKB_Swiss-Prot
      program: mmseqs

KOFamScan:
  # Number of threads task will use
  threads: 1
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  skip: true
  dependencies:
    KofamscanExecAnnotation:
      program: /path/To/kofamscan/exec_annotation
      kolist: /path/To/kofam/ko_list
      profiles: /path/To/profiles/eukaryote.hal

EggNog:
  # Number of threads task will use
  threads: 1
  # Amount of memory task will use (in GB)
  memory: 8
  time: "4:00:00"
  skip: true
  dependencies:
    EMapper:
      program: emapper.py
      FLAGS:
        # Provide flags as needed
        -m diamond
        --override
        --data_dir /location/of/eggnog-data

...  # document end

Call pipeline

Activate your EukMS_report conda environment.

conda activate EukMS_report

Run pipeline using the command:

yapim run -i /path/to/EukMetaSanity/tests/data -c report-config.yaml -p $EukMS_report

Note that we do not need to provide the input directory for this analysis, as the pipeline will only annotate genomes that have completed the Run or Refine pipeline.

With default settings, the analysis should complete in less than 10 minutes.

Report output

This will update the directory structure:

out/
  |-- wdir/
  |-- input/
  |-- report-eukmetasanity.log
  |-- refine-eukmetasanity.log
  |-- run-eukmetasanity.log
  |-- results/
      |-- report/
          |-- report.pkl
          |-- NAO-all-DCM-20-180-00_bin-1/
              ... (results based on annotation programs run)
          |-- NAO-all-DCM-20-180-00_bin-19/
              ...
          |-- NAO-all-DCM-20-180-00_bin-2/
              ...
      |-- refine/
          ...
      |-- run/
          ...

Citations

"The high-throughput gene prediction of more than 1,700 eukaryote genomes using the software package EukMetaSanity" by Neely, Hu, Alexander, and Tully, 2021.

Also cite all dependencies that you used, as EukMetaSanity would not be possible were it not for the developers of these programs.

(citation list upcoming)