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Snakefile
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###########################################################################
# Genome annotation pipeline using Maker in Snakemake
# Snakemake/5.13.0
###########################################################################
from os.path import join
from snakemake.io import expand, glob_wildcards
from Bio import SeqIO
# Load configuration paths and files
result_dir = config["result_dir"] # Directory to store results
input_dir = config["input_dir"] # Directory containing input files
repeat_file = config["repeat_file"] # File containing repeat sequences
protein_file = config["protein_file"] # Protein reference file(s)
transcript_file = config["transcript_file"] # Transcript reference file(s)
augustus = config["augustus"] # Augustus configuration file
SAMPLE = list(glob_wildcards(join(input_dir, "{ids}.fasta")))[0]
if not SAMPLE:
raise ValueError("No FASTA files found in the input directory!")
PROT = list(protein_file.split(",")) # Split multiple protein files
PROT_NAME = [os.path.basename(x) for x in PROT]
PROT_NAME = [x.rstrip(".fasta").rstrip(".faa") for x in PROT_NAME]
print(SAMPLE)
print(PROT_NAME)
rule All:
input:
# Repeats
expand(join(input_dir,"{samples}-families.fa"),samples=SAMPLE),
expand(join(result_dir,"{samples}-families.fa"),samples=SAMPLE),
expand(join(result_dir,"{samples}.fasta"),samples=SAMPLE),
expand(join(result_dir,"{samples}.fasta.masked"),samples=SAMPLE),
expand(join(result_dir,"{samples}.fasta.out.gff"),samples=SAMPLE),
# Maker ctrl files
expand(join(result_dir,"{samples}.maker_opts_rnd1.ctl"),samples=SAMPLE),
expand(join(result_dir,"{samples}.maker_opts_rnd2.ctl"),samples=SAMPLE),
expand(join(result_dir,"{samples}.maker_opts_rnd3.ctl"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1_master_datastore_index.log"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2_master_datastore_index.log"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3_master_datastore_index.log"),samples=SAMPLE),
# Maker GFFs
expand(join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1.all.gff"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd1.maker.output/snap/rnd1.snap.hmm"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.gff"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.gff"),samples=SAMPLE),
# rename gffs
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.gff3"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.gtf"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.clean.gtf"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.fna"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.faa"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.gff3"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.gtf"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.clean.gtf"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.fna"),samples=SAMPLE),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.faa"),samples=SAMPLE),
# functional annotation
expand(join(result_dir, "{samples}.rnd2.maker.output/{samples}.{prots}.gff3"),samples=SAMPLE,prots=PROT_NAME),
expand(join(result_dir,"{samples}.rnd2.maker.output/{samples}.{prots}.blast"),samples=SAMPLE,prots=PROT_NAME),
expand(join(result_dir, "{samples}.rnd3.maker.output/{samples}.{prots}.gff3"),samples=SAMPLE,prots=PROT_NAME),
expand(join(result_dir,"{samples}.rnd3.maker.output/{samples}.{prots}.blast"),samples=SAMPLE,prots=PROT_NAME),
rule RepeatModeler:
input:
fa=join(input_dir,"{samples}.fasta"),
output:
fa=join(input_dir,"{samples}-families.fa"),
fa2=join(result_dir,"{samples}.fasta"),
rep=join(result_dir,"{samples}-families.fa"),
params:
rname="RepeatModeler",
dir=input_dir,
id="{samples}",
opdir=result_dir,
threads:
48
shell:
"""
mkdir -p {params.opdir}
cd {params.dir}
module load repeatmodeler
BuildDatabase -name {params.id} {input.fa}
RepeatModeler -database {params.id} -pa {threads} -LTRStruct >& {params.id}.out
cp {output.fa} {output.rep}
cp {input.fa} {output.fa2}
"""
rule RepeatMasker:
input:
fa=join(result_dir,"{samples}.fasta"),
rep=join(result_dir,"{samples}-families.fa"),
output:
fa=join(result_dir,"{samples}.fasta.masked"),
gff=join(result_dir,"{samples}.fasta.out.gff"),
params:
rname="RepeatMasker",
dir=result_dir,
#rep=repeat_file,
threads="48",
shell:
"""
cd {params.dir}
module load repeatmasker
RepeatMasker -u -s -poly -engine rmblast -pa {params.threads} -gff -no_is -gccalc -norna -lib {input.rep} {input.fa}
"""
rule maker_opts1:
input:
fa=join(result_dir,"{samples}.fasta.masked"),
output:
ctl=join(result_dir,"{samples}.maker_opts_rnd1.ctl"),
params:
rname="maker_opts1",
protein=protein_file,
transcript=transcript_file,
augustus=augustus,
scripts_path=join(result_dir,"param_files"),
outdir=result_dir,
shell:
"""
mkdir -p {params.outdir}
python3 {params.scripts_path}/generate_opts1.py {output.ctl} {input.fa} {params.protein} {params.transcript} {params.augustus}
"""
rule maker_rnd1:
input:
fa=join(result_dir,"{samples}.fasta.masked"),
ctl=join(result_dir,"{samples}.maker_opts_rnd1.ctl"),
output:
log=join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1_master_datastore_index.log"),
params:
rname="maker_rnd1",
outid="{samples}.rnd1",
outdir=result_dir,
bopts=join(result_dir,"param_files/maker_bopts.log"),
exe=join(result_dir,"param_files/maker_exe.log"),
shell:
"""
module load maker
cd {params.outdir}
valgrind mpiexec -c 1 -np 40 maker -RM_off -base {params.outid} {input.ctl} {params.bopts} {params.exe}
"""
rule make_gff1:
input:
log=join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1_master_datastore_index.log"),
output:
gff=join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1.all.gff"),
snap=join(result_dir,"{samples}.rnd1.maker.output/snap/rnd1.snap.hmm"),
params:
rname="make_gff1",
outdir=result_dir,
shell:
"""
module load maker
cd {params.outdir}/{wildcards.samples}.rnd1.maker.output/
gff3_merge -d {input.log}
mkdir -p {params.outdir}/{wildcards.samples}.rnd1.maker.output/snap
cd {params.outdir}/{wildcards.samples}rnd1.maker.output/snap
maker2zff -x 0.5 -l 50 -c 0 -e 0 -o 0 -d {input.log}
fathom genome.ann genome.dna -gene-stats > gene-stats.log
fathom genome.ann genome.dna -validate > validate.log
fathom genome.ann genome.dna -categorize 1000 > categorize.log
fathom uni.ann uni.dna -export 1000 -plus > uni-plus.log
mkdir -p params
cd params
forge ../export.ann ../export.dna > ../forge.log
cd ..
hmm-assembler.pl genome params > {output.snap}
"""
rule maker_opts2:
input:
fa=join(result_dir,"{samples}.fasta.masked"),
gff=join(result_dir,"{samples}.rnd1.maker.output/{samples}.rnd1.all.gff"),
snap=join(result_dir,"{samples}.rnd1.maker.output/snap/rnd1.snap.hmm"),
output:
ctl2=join(result_dir,"{samples}.maker_opts_rnd2.ctl"),
ctl3=join(result_dir,"{samples}.maker_opts_rnd3.ctl"),
params:
rname="maker_opts2",
protein=protein_file,
transcript=transcript_file,
scripts_path=join(result_dir,"param_files"),
outdir=result_dir,
shell:
"""
mkdir -p {params.outdir}
python3 {params.scripts_path}/generate_opts2.py {output.ctl2} {input.fa} {input.gff} {input.snap} {params.protein} {params.transcript}
python3 {params.scripts_path}/generate_opts3.py {output.ctl3} {input.fa} {input.gff} {input.snap} {params.protein} {params.transcript}
"""
rule maker_rnd2:
input:
fa=join(result_dir,"{samples}.fasta.masked"),
ctl=join(result_dir,"{samples}.maker_opts_rnd2.ctl"),
output:
log=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2_master_datastore_index.log"),
params:
rname="maker_rnd2",
outid="{samples}.rnd2",
outdir=result_dir,
bopts=join(result_dir,"param_files/maker_bopts.log"),
exe=join(result_dir,"param_files/maker_exe.log"),
shell:
"""
module load maker
cd {params.outdir}
valgrind mpiexec -c 1 -np 40 maker -RM_off -base {params.outid} {input.ctl} {params.bopts} {params.exe}
"""
rule make_gff2:
input:
log=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2_master_datastore_index.log"),
fa=join(result_dir,"{samples}.fasta"),
output:
gff=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.gff"),
faa=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.faa"),
fna=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.fna"),
params:
rname="make_gff2",
outdir=result_dir
shell:
"""
module load maker snap
cd {params.outdir}/{wildcards.samples}.rnd2.maker.output/
gff3_merge -d {input.log}
/data/OpenOmics/references/brakerMake/gffread/gffread -g {input.fa} -y {output.faa} -x {output.fna} {output.gff}
"""
rule rnd2_gff_rename:
input:
gff=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.gff"),
aa=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.faa"),
cds=join(result_dir,"{samples}.rnd2.maker.output/{samples}.rnd2.all.fna"),
output:
map=temp(join(result_dir, "{samples}.rnd2.maker.output/{samples}.map")),
gff=join(result_dir, "{samples}.rnd2.maker.output/{samples}.gff3"),
aa=join(result_dir, "{samples}.rnd2.maker.output/{samples}.faa"),
cds=join(result_dir, "{samples}.rnd2.maker.output/{samples}.fna"),
params:
species_id="{samples}",
rname="rnd2_gff_rename",
shell:
"""
module load maker
maker_map_ids --prefix {params.species_id} --justify 5 {input.gff} > {output.map}
cp {input.gff} {output.gff}
map_gff_ids {output.map} {output.gff}
cp {input.aa} {output.aa}
cp {input.cds} {output.cds}
map_fasta_ids {output.map} {output.aa}
map_fasta_ids {output.map} {output.cds}
"""
rule rnd2_gff2gtf:
input:
gff=join(result_dir, "{samples}.rnd2.maker.output/{samples}.gff3"),
output:
gtf=join(result_dir, "{samples}.rnd2.maker.output/{samples}.gtf"),
clean=join(result_dir, "{samples}.rnd2.maker.output/{samples}.clean.gtf"),
params:
rname="rnd2_gff2gtf",
shell:
"""
module load agat/1.2.0 python
agat_convert_sp_gff2gtf.pl --gff {input.gff} -o {output.gtf}
python /data/OpenOmics/references/brakerMake/clean_gtf.py {output.gtf} > {output.clean}
"""
rule rnd2_gff_annot:
input:
gff=join(result_dir, "{samples}.rnd2.maker.output/{samples}.gff3"),
prot=join(result_dir, "{samples}.rnd2.maker.output/{samples}.faa"),
cds=join(result_dir, "{samples}.rnd2.maker.output/{samples}.fna"),
output:
gff=join(result_dir, "{samples}.rnd2.maker.output/{samples}.{prots}.gff3"),
blast=join(result_dir,"{samples}.rnd2.maker.output/{samples}.{prots}.blast"),
params:
rname="rnd2_gff_annot",
threads=8,
uniprot=protein_file,
shell:
"""
module load blast maker
blastp -query {input.prot} -db {params.uniprot} -evalue 1e-6 -max_hsps 1 -max_target_seqs 1 -outfmt 6 -out {output.blast} -num_threads {params.threads}
maker_functional_gff {params.uniprot} {output.blast} {input.gff} > {output.gff}
"""
rule maker_rnd3:
input:
fa=join(result_dir,"{samples}.fasta.masked"),
ctl=join(result_dir,"{samples}.maker_opts_rnd3.ctl"),
output:
log=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3_master_datastore_index.log"),
params:
rname="maker_rnd3",
outid="{samples}.rnd3",
outdir=result_dir,
bopts=join(result_dir,"param_files/maker_bopts.log"),
exe=join(result_dir,"param_files/maker_exe.log"),
shell:
"""
module load maker
cd {params.outdir}
valgrind mpiexec -c 1 -np 40 maker -RM_off -base {params.outid} {input.ctl} {params.bopts} {params.exe}
"""
rule make_gff3:
input:
log=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3_master_datastore_index.log"),
fa=join(result_dir,"{samples}.fasta"),
output:
gff=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.gff"),
faa=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.faa"),
fna=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.fna"),
params:
rname="make_gff3",
outdir=result_dir,
shell:
"""
module load maker snap
cd {params.outdir}/{wildcards.samples}.rnd3.maker.output/
gff3_merge -d {input.log}
/data/OpenOmics/references/brakerMake/gffread/gffread -g {input.fa} -y {output.faa} -x {output.fna} {output.gff}
"""
rule rnd3_gff_rename:
input:
gff=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.gff"),
aa=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.faa"),
cds=join(result_dir,"{samples}.rnd3.maker.output/{samples}.rnd3.all.fna"),
output:
map=temp(join(result_dir, "{samples}.rnd3.maker.output/{samples}.map")),
gff=join(result_dir, "{samples}.rnd3.maker.output/{samples}.gff3"),
aa=join(result_dir, "{samples}.rnd3.maker.output/{samples}.faa"),
cds=join(result_dir, "{samples}.rnd3.maker.output/{samples}.fna"),
params:
species_id="{samples}",
rname="rnd3_gff_rename",
shell:
"""
module load maker
maker_map_ids --prefix {params.species_id} --justify 5 {input.gff} > {output.map}
cp {input.gff} {output.gff}
map_gff_ids {output.map} {output.gff}
cp {input.aa} {output.aa}
cp {input.cds} {output.cds}
map_fasta_ids {output.map} {output.aa}
map_fasta_ids {output.map} {output.cds}
"""
rule rnd3_gff2gtf:
input:
gff=join(result_dir, "{samples}.rnd3.maker.output/{samples}.gff3"),
output:
gtf=join(result_dir, "{samples}.rnd3.maker.output/{samples}.gtf"),
clean=join(result_dir, "{samples}.rnd3.maker.output/{samples}.clean.gtf"),
params:
rname="rnd3_gff2gtf",
shell:
"""
module load agat/1.2.0 python
agat_convert_sp_gff2gtf.pl --gff {input.gff} -o {output.gtf}
python /data/OpenOmics/references/brakerMake/clean_gtf.py {output.gtf} > {output.clean}
"""
rule rnd3_gff_annot:
input:
gff=join(result_dir, "{samples}.rnd3.maker.output/{samples}.gff3"),
prot=join(result_dir, "{samples}.rnd3.maker.output/{samples}.faa"),
cds=join(result_dir, "{samples}.rnd3.maker.output/{samples}.fna"),
output:
gff=join(result_dir, "{samples}.rnd3.maker.output/{samples}.{prots}.gff3"),
blast=join(result_dir,"{samples}.rnd3.maker.output/{samples}.{prots}.blast"),
params:
rname="rnd3_gff_annot",
threads=8,
uniprot=protein_file,
shell:
"""
module load blast maker
blastp -query {input.prot} -db {params.uniprot} -evalue 1e-6 -max_hsps 1 -max_target_seqs 1 -outfmt 6 -out {output.blast} -num_threads {params.threads}
maker_functional_gff {params.uniprot} {output.blast} {input.gff} > {output.gff}
"""