Here it goes, a massive undertaking of attempting to annotate the Pgenerosa_v070 genome (FastA; 2.1GB) using MAKER on Mox! I previously started this on 20190115, but killed it in order to fix a number of different issues with the script that were causing problems. I decided the changes were substantial enough, that I’d just make a new working directory and notebook entry.

This will perform the following:

one round of MAKER gene model predictions
two rounds of SNAP gene model training/predictions
renaming of gene models to NCBI-standardized convention
functional characterization of protein models (via BLASTp)
functional characterization of protein domains (via InterProScan5)

It also takes a slew of input files! See the SBATCH script and the list below for those:

Genome assembly

Pgenerosa_v070.fa

Transcriptome assembly (from 20180904):

20180827_trinity_geoduck.fasta (972MB)

Protein FastA (from 20181121)

20180827_trinity_geoduck.fasta.transdecoder.pep (142MB)

Repeats library FastA (from 20181219)

Pgenerosa_v070-families.fa (1.4MB)

I also annotated a subset of this genome (Pgenerosa_v071; scaffolds >10kbp) on 20190213](https://robertslab.github.io/sams-notebook/posts/2019/2019-02-13-Genome-Annotation—Pgenerosa_v71-with-MAKER-on-Mox/). Just putting that in here for reference purposes.

SBATCH script (GitHub):

20190228_pgen_maker_v070_annotation.sh


#!/bin/bash
## Job Name
#SBATCH --job-name=maker
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=2
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=25-00:00:00
## Memory per node
#SBATCH --mem=120G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --workdir=/gscratch/scrubbed/samwhite/outputs/20190228_pgen_maker_v070_annotation

# Exit if any command fails
set -e

# Load Python Mox module for Python module availability

module load intel-python3_2017

# Load Open MPI module for parallel, multi-node processing

module load icc_19-ompi_3.1.2

# SegFault fix?
export THREADS_DAEMON_MODEL=1

# Document programs in PATH (primarily for program version ID)

date >> system_path.log
echo "" >> system_path.log
echo "System PATH for $SLURM_JOB_ID" >> system_path.log
echo "" >> system_path.log
printf "%0.s-" {1..10} >> system_path.log
echo ${PATH} | tr : \\n >> system_path.log

# Add BLAST to system PATH
export PATH=$PATH:/gscratch/srlab/programs/ncbi-blast-2.6.0+/bin
export BLASTDB=/gscratch/srlab/blastdbs/UniProtKB_20181008


## Establish variables for more readable code

wd=$(pwd)
maker_dir=/gscratch/srlab/programs/maker-2.31.10/bin
snap_dir=/gscratch/srlab/programs/maker-2.31.10/exe/snap

### Paths to Maker binaries

maker=${maker_dir}/maker
gff3_merge=${maker_dir}/gff3_merge
maker2zff=${maker_dir}/maker2zff
fathom=${snap_dir}/fathom
forge=${snap_dir}/forge
hmmassembler=${snap_dir}/hmm-assembler.pl
fasta_merge=${maker_dir}/fasta_merge
map_ids=${maker_dir}/maker_map_ids
map_gff_ids=${maker_dir}/map_gff_ids
map_fasta_ids=${maker_dir}/map_fasta_ids
functional_fasta=${maker_dir}/maker_functional_fasta
functional_gff=${maker_dir}/maker_functional_gff
ipr_update_gff=${maker_dir}/ipr_update_gff
iprscan2gff3=${maker_dir}/iprscan2gff3

blastp_dir=${wd}/blastp_annotation
maker_blastp=${wd}/blastp_annotation/blastp.outfmt6
maker_prot_fasta=${wd}/snap02/Pgenerosa_v070_snap02.all.maker.proteins.fasta
maker_prot_fasta_renamed=${wd}/snap02/Pgenerosa_v070_snap02.all.maker.proteins.renamed.fasta
maker_transcripts_fasta=${wd}/snap02/Pgenerosa_v070_snap02.all.maker.transcripts.fasta
maker_transcripts_fasta_renamed=${wd}/snap02/Pgenerosa_v070_snap02.all.maker.transcripts.renamed.fasta
snap01_est_gff=${wd}/snap01/Pgenerosa_v070_snap01.maker.all.noseqs.est2genome.gff
snap02_gff=${wd}/snap02/Pgenerosa_v070_snap02.all.gff
snap02_gff_renamed=${wd}/snap02/Pgenerosa_v070_snap02.all.renamed.gff
snap01_protein_gff=${wd}/snap01/Pgenerosa_v070_snap01.maker.all.noseqs.protein2genome.gff
snap01_rm_gff=${wd}/snap01/Pgenerosa_v070_snap01.maker.all.noseqs.repeats.gff
put_func_gff=Pgenerosa_v070_genome_snap02.all.renamed.putative_function.gff
put_func_prot=Pgenerosa_v070_genome_snap02.all.maker.proteins.renamed.putative_function.fasta
put_func_trans=Pgenerosa_v070_genome_snap02.all.maker.transcripts.renamed.putative_function.fasta
put_domain_gff=Pgenerosa_v070_genome_snap02.all.renamed.putative_function.domain_added.gff
ips_dir=${wd}/interproscan_annotation
ips_base=Pgenerosa_v070_maker_proteins_ips
ips_name=Pgenerosa_v070_maker_proteins_ips.tsv
id_map=${wd}/snap02/Pgenerosa_v070_genome.map
ips_domains=Pgenerosa_v070_genome_snap02.all.renamed.visible_ips_domains.gff

## Path to blastp
blastp=/gscratch/srlab/programs/ncbi-blast-2.6.0+/bin/blastp

## Path to InterProScan5
interproscan=/gscratch/srlab/programs/interproscan-5.31-70.0/interproscan.sh

## Store path to options control file
maker_opts_file=./maker_opts.ctl

### Path to genome FastA file
genome=/gscratch/srlab/sam/data/P_generosa/generosa_genomes/Pgenerosa_v070.fa

### Path to transcriptome FastA file
transcriptome=/gscratch/srlab/sam/data/P_generosa/generosa_transcriptomes/20180827_trinity_geoduck.fasta

### Path to Crassotrea gigas NCBI protein FastA
gigas_proteome=/gscratch/srlab/sam/data/C_gigas/gigas_ncbi_protein/GCA_000297895.1_oyster_v9_protein.faa

### Path to Crassostrea virginica NCBI protein FastA
virginica_proteome=/gscratch/srlab/sam/data/C_virginica/virginica_ncbi_protein/GCF_002022765.2_C_virginica-3.0_protein.faa

### Path to Panopea generosa TransDecoder protein FastA
panopea_td_proteome=/gscratch/srlab/sam/data/P_generosa/generosa_proteomes/20180827_trinity_geoduck.fasta.transdecoder.pep

### Path to concatenated NCBI prteins FastA
combined_proteomes=${wd}/combined_proteomes.fasta

### Path to P.generosa-specific repeat library
repeat_library=/gscratch/srlab/sam/data/P_generosa/generosa_repeats/Pgenerosa_v070-families.fa

### Path to SwissProt database for BLASTp
sp_db_blastp=/gscratch/srlab/blastdbs/UniProtKB_20190109/uniprot_sprot.fasta


## Make directories
mkdir blastp_annotation
mkdir interproscan_annotation
mkdir snap01
mkdir snap02


## Create Maker control files needed for running Maker, only if it doesn't already exist and then edit it.
### Edit options file
### Set paths to P.generosa genome and transcriptome.
### Set path to combined C. gigas, C.virginica, P.generosa proteomes.
### The use of the % symbol sets the delimiter sed uses for arguments.
### Normally, the delimiter that most examples use is a slash "/".
### But, we need to expand the variables into a full path with slashes, which screws up sed.
### Thus, the use of % symbol instead (it could be any character that is NOT present in the expanded variable; doesn't have to be "%").
if [ ! -e maker_opts.ctl ]; then
  $maker -CTL
  sed -i "/^genome=/ s% %$genome %" "$maker_opts_file"
  sed -i "/^est=/ s% %$transcriptome %" "$maker_opts_file"
  sed -i "/^protein=/ s% %$combined_proteomes %" "$maker_opts_file"
  sed -i "/^rmlib=/ s% %$repeat_library %" "$maker_opts_file"
  sed -i "/^est2genome=0/ s/est2genome=0/est2genome=1/" "$maker_opts_file"
  sed -i "/^protein2genome=0/ s/protein2genome=0/protein2genome=1/" "$maker_opts_file"
fi

## Create combined proteome FastA file, only if it doesn't already exist.
if [ ! -e combined_proteomes.fasta ]; then
    touch combined_proteomes.fasta
    cat "$gigas_proteome" >> combined_proteomes.fasta
    cat "$virginica_proteome" >> combined_proteomes.fasta
    cat "$panopea_td_proteome" >> combined_proteomes.fasta
fi



## Run Maker
### Specify number of nodes to use.
mpiexec -n 56 $maker

## Merge gffs
${gff3_merge} -d Pgenerosa_v070.maker.output/Pgenerosa_v070_master_datastore_index.log

## GFF with no FastA in footer
${gff3_merge} -n -s -d Pgenerosa_v070.maker.output/Pgenerosa_v070_master_datastore_index.log > Pgenerosa_v070.maker.all.noseqs.gff

## Merge all FastAs
${fasta_merge} -d Pgenerosa_v070.maker.output/Pgenerosa_v070_master_datastore_index.log

## Extract GFF alignments for use in subsequent MAKER rounds
### Transcript alignments
awk '{ if ($2 == "est2genome") print $0 }' Pgenerosa_v070.maker.all.noseqs.gff > Pgenerosa_v070.maker.all.noseqs.est2genome.gff
### Protein alignments
awk '{ if ($2 == "protein2genome") print $0 }' Pgenerosa_v070.maker.all.noseqs.gff > Pgenerosa_v070.maker.all.noseqs.protein2genome.gff
### Repeat alignments
awk '{ if ($2 ~ "repeat") print $0 }' Pgenerosa_v070.maker.all.noseqs.gff > Pgenerosa_v070.maker.all.noseqs.repeats.gff

## Run SNAP training, round 1
cd ${wd}
cd snap01
${maker2zff} ../Pgenerosa_v070.all.gff
${fathom} -categorize 1000 genome.ann genome.dna
${fathom} -export 1000 -plus uni.ann uni.dna
${forge} export.ann export.dna
${hmmassembler} genome . > Pgenerosa_v070_snap01.hmm

## Initiate second Maker run.
### Copy initial maker control files and
### - change gene prediction settings to 0 (i.e. don't generate Maker gene predictions)
### - use GFF subsets generated in first round of MAKER
### - set location of snaphmm file to use for gene prediction
### Percent symbols used below are the sed delimiters, instead of the default "/",
### due to the need to use file paths.
if [ ! -e maker_opts.ctl ]; then
  $maker -CTL
  sed -i "/^genome=/ s% %$genome %" maker_opts.ctl
  sed -i "/^est2genome=1/ s/est2genome=1/est2genome=0/" maker_opts.ctl
  sed -i "/^protein2genome=1/ s/protein2genome=1/protein2genome=0/" maker_opts.ctl
  sed -i "/^est_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.est2genome.gff %" maker_opts.ctl
  sed -i "/^protein_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.protein2genome.gff %" maker_opts.ctl
  sed -i "/^rm_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.repeats.gff %" maker_opts.ctl
  sed -i "/^snaphmm=/ s% %Pgenerosa_v070_snap01.hmm %" maker_opts.ctl
fi

## Run Maker
### Set basename of files and specify number of CPUs to use
mpiexec -n 56 $maker \
-base Pgenerosa_v070_snap01

## Merge gffs
${gff3_merge} -d Pgenerosa_v070_snap01.maker.output/Pgenerosa_v070_snap01_master_datastore_index.log

## GFF with no FastA in footer
${gff3_merge} -n -s -d Pgenerosa_v070_snap01.maker.output/Pgenerosa_v070_snap01_master_datastore_index.log > Pgenerosa_v070_snap01.maker.all.noseqs.gff

## Run SNAP training, round 2
cd ${wd}
cd snap02
${maker2zff} ../snap01/Pgenerosa_v070_snap01.all.gff
${fathom} -categorize 1000 genome.ann genome.dna
${fathom} -export 1000 -plus uni.ann uni.dna
${forge} export.ann export.dna
${hmmassembler} genome . > Pgenerosa_v070_snap02.hmm

## Initiate third and final Maker run.

if [ ! -e maker_opts.ctl ]; then
  $maker -CTL
  sed -i "/^genome=/ s% %$genome %" maker_opts.ctl
  sed -i "/^est2genome=1/ s/est2genome=1/est2genome=0/" maker_opts.ctl
  sed -i "/^protein2genome=1/ s/protein2genome=1/protein2genome=0/" maker_opts.ctl
  sed -i "/^est_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.est2genome.gff %" maker_opts.ctl
  sed -i "/^protein_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.protein2genome.gff %" maker_opts.ctl
  sed -i "/^rm_gff=/ s% %../Pgenerosa_v070.maker.all.noseqs.repeats.gff %" maker_opts.ctl
  sed -i "/^snaphmm=/ s% %Pgenerosa_v070_snap02.hmm %" maker_opts.ctl
fi

## Run Maker
### Set basename of files and specify number of CPUs to use
mpiexec -n 56 $maker \
-base Pgenerosa_v070_snap02

## Merge gffs
${gff3_merge} \
-d Pgenerosa_v070_snap02.maker.output/Pgenerosa_v070_snap02_master_datastore_index.log

## GFF with no FastA in footer
${gff3_merge} -n -s -d Pgenerosa_v070_snap02.maker.output/Pgenerosa_v070_snap02_master_datastore_index.log > Pgenerosa_v070_snap02.maker.all.noseqs.gff

## Merge FastAs
${fasta_merge} \
-d Pgenerosa_v070_snap02.maker.output/Pgenerosa_v070_snap02_master_datastore_index.log

# Create copies of files for mapping
cp ${maker_prot_fasta} ${maker_prot_fasta_renamed}
cp ${maker_transcripts_fasta} ${maker_transcripts_fasta_renamed}
cp ${snap02_gff} ${snap02_gff_renamed}

# Map IDs
## Change gene names
${map_ids} \
--prefix PGEN_ \
--justify 8 \
${snap02_gff} \
> ${id_map}

## Map GFF IDs
${map_gff_ids} \
${id_map} \
${snap02_gff_renamed}

## Map FastAs
### Proteins
${map_fasta_ids} \
${id_map} \
${maker_prot_fasta_renamed}

### Transcripts
${map_fasta_ids} \
${id_map} \
${maker_transcripts_fasta_renamed}

# Run InterProScan 5
## disable-precalc since this requires external database access (which Mox does not allow)
cd ${ips_dir}

${interproscan} \
--input ${maker_prot_fasta_renamed} \
--goterms \
--output-file-base ${ips_base} \
--disable-precalc

# Run BLASTp
cd ${blastp_annotation}

${blastp} \
-query ${maker_prot_fasta_renamed} \
-db ${sp_db_blastp} \
-out ${maker_blastp} \
-max_target_seqs 1 \
-evalue 1e-6 \
-outfmt 6 \
-num_threads 28


# Functional annotations

cd ${wd}

## Add putative gene functions
### GFF
${functional_gff} \
${sp_db_blastp} \
${maker_blastp} \
${snap02_gff_renamed} \
> ${put_func_gff}

### Proteins
${functional_fasta} \
${sp_db_blastp} \
${maker_blastp} \
${maker_prot_fasta_renamed} \
> ${put_func_prot}

### Transcripts
${functional_fasta} \
${sp_db_blastp} \
${maker_blastp} \
${maker_transcripts_fasta_renamed} \
> ${put_func_trans}

## Add InterProScan domain info
### Add searchable tags
${ipr_update_gff} \
${put_func_gff} \
${ips_dir}/${ips_name} \
> ${put_domain_gff}

### Add viewable features for genome browsers (JBrowse, Gbrowse, Web Apollo)
${iprscan2gff3} \
${ips_dir}/${ips_name} \
${snap02_gff_renamed} \
> ${ips_domains}

RESULTS

Well, it finally finished! Praise be! Cumulative actual runtime was ~36 days! However, the job was interrupted a few times (intentionally for changes and unintentionally for Mox maintenance junk). The beauty of MAKER is that it will continue where it left off. Without this feature, using it in our current HPC configuration would probably be a non-starter.

Pgenerosa_v070 cumulative runtimes screencap

Output folder:

20190228_pgen_maker_v070_annotation/

The important files:

Pgenerosa_v070_genome_snap02.all.renamed.putative_function.domain_added.gff (7.1GB)
- GFF file with all contigs annotated with putative functions and functional domains.
- INCLUDES SEQUENCE FASTAS AT END OF FILE!
- Generated with one round of MAKER gene prediction, followed by two rounds of SNAP ab-initio gene prediction.
- MD5: ba6e9c69951d0b71675e3dc471563a0e
Pgenerosa_v070_genome_snap02.all.maker.proteins.renamed.putative_function.fasta (17MB)
- Annotated proteins FastA file.
- Generated with one round of MAKER gene prediction, followed by two rounds of SNAP ab-initio gene prediction.
Pgenerosa_v070_genome_snap02.all.maker.transcripts.renamed.putative_function.fasta (44MB)
- Annotated proteins FastA file.
- Generated with one round of MAKER gene prediction, followed by two rounds of SNAP ab-initio gene prediction.

I’ll make an additional post that delves into more of these results, breaks out different genome structures into separate GFF files (e.g. exons, mRNA, CDS, etc.), and compares them to the other annotation performed on the 10kbp genome subset. However, a quick grep -c ">" on the FastA files reveals:

53035 annotated transcripts/proteins