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species_workflow.py
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'''
------------------------------------------------------------------------------------------------------------------------
This workflow is used to transform the raw sequence data into sequences ready for alignment.
Workflow is the following
1: Secapr quality check of the sequences
2: Trimming of the sequences using trimmomatic
3: Hybpipering the species in order to create the exons of the baits for each species
4: Checking for Paralogs
5: Running Intronerate again to get the Introns for each species
6: Trimming for Coverage of sequencing and joining the exon and intron of each species to create supercontigs
------------------------------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------------------------------------
Author: Oscar Wrisberg
Date: 10/11/2021
------------------------------------------------------------------------------------------------------------------------
'''
from os import O_SYNC, name
from gwf import Workflow, AnonymousTarget
import os.path
from inspect import getsourcefile
# import math
# import glob
gwf = Workflow()
########################################################################################################################
################################################---- Renaming the sequences ----#######################################
########################################################################################################################
def renamer(path_in ,rename_file, done,remove_file):
""""""
inputs = []
outputs = [done]
options = {'cores': 1, 'memory': "10g", 'walltime': "00:10:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
# Use the names_1_data_1_rename_seqs.csv to rename the sequences in folder
cd {path_in}
bash /home/owrisberg/Coryphoideae/github_code/renamer_remove.sh -r {rename_file} -d {remove_file}
""".format(path_in = path_in, done = done, rename_file = rename_file, remove_file = remove_file)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
################################################---- Fastqc quality check raw ----#######################################
########################################################################################################################
def fastqc_raw(species,path_in ,path_out, done,):
"""Quality checking using fastqc as this should work on individual species"""
inputs = []
outputs = [path_out+species+"_R1_fastqc.html",path_out+species+"_R2_fastqc.html", done]
options = {'cores': 1, 'memory': "10g", 'walltime': "00:30:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate secapr_env
fastqc -o {output} {path_in}{species}_R1.fastq {path_in}{species}_R2.fastq
echo touching {done}
touch {done}
""".format(path_in = path_in,species = species, output = path_out, done = done)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
################################################---- Fastqc quality check trimmed ----#######################################
########################################################################################################################
def fastqc_trimmed(species,path_in ,path_out, done,):
"""Quality checking using fastqc as this should work on individual species"""
inputs = [path_in+species+"_UN.fastq", path_in+species+"_1PU.fastq", path_in+species+"_2PU.fastq","/home/owrisberg/Coryphoideae/work_flow/02_trimmed/done/"+species]
outputs = [path_out+species+"_1PU_fastqc.html", path_out+species+"_2PU_fastqc.html",path_out+species+"_UN_fastqc.html" ,done]
options = {'cores': 1, 'memory': "10g", 'walltime': "00:30:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate secapr_env
fastqc -o {output} {path_in}{species}_1PU.fastq {path_in}{species}_2PU.fastq {path_in}{species}_UN.fastq
touch {done}
""".format(path_in = path_in,species = species, output = path_out, done = done)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
# ########################################################################################################################
# ################################################---- Trimmomatic ----###################################################
# ########################################################################################################################
def trimmomatic(species, path_in, path_out, done):
"""Trimming raw data using trimmomatic with custom adapter.
Afterwards combines paired and unpaired reads for forward and reverse reads respectively for each species
to enable post-trimming secapr quality_check for comparability before and after trimming """
inputs = []
outputs = [path_out+species+"_UN.fastq",path_out+"secapr_postrim/"+species+"_UN.fastq", done, path_out+species+"_1P.fastq", path_out+species+"_2P.fastq"]
options = {'cores': 16, 'memory': "10g", 'walltime': "01:00:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate trimmomatic_env
trimmomatic PE -threads 16 -phred33 {input}_R1.fastq {input}_R2.fastq -baseout {output}.fastq\
ILLUMINACLIP:/home/owrisberg/miniconda3/pkgs/trimmomatic-0.39-1/share/trimmomatic-0.39-1/adapters/TruSeq3-PE-2.fa:2:30:10:1:true\
LEADING:3\
TRAILING:3\
MAXINFO:40:0.8\
MINLEN:36\
2>> stderr_trim_loop_output.txt
echo combining {path_out}{species}_1P.fastq and {path_out}{species}_1U.fastq into {path_out}secapr_postrim/{species}_1PU.fastq
cat {path_out}{species}_1P.fastq {path_out}{species}_1U.fastq > {path_out}secapr_postrim/{species}_1PU.fastq
echo combining {path_out}{species}_2P.fastq and {path_out}{species}_2U.fastq into {path_out}secapr_postrim/{species}_2PU.fastq
cat {path_out}{species}_2P.fastq {path_out}{species}_2U.fastq > {path_out}secapr_postrim/{species}_2PU.fastq
echo combining {path_out}{species}_1U.fastq {path_out}{species}_2U.fastq > {path_out}{species}_UN.fastq
cat {path_out}{species}_1U.fastq {path_out}{species}_2U.fastq > {path_out}{species}_UN.fastq
cp {path_out}{species}_UN.fastq {path_out}secapr_postrim/
echo Removing {path_out}{species}_1U.fastq
rm {path_out}{species}_1U.fastq
echo Removing {path_out}{species}_2U.fastq
rm {path_out}{species}_2U.fastq
touch {done}
""".format(input = path_in + species, output = path_out+species, done = done, species = species, path_out = path_out)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
#########################################---- Create a namelist.txt ----################################################
########################################################################################################################
def namelist_function(path_in, path_out, done):
"""Create a namelist of all the species in the trimmomatic folder."""
inputs = [] # The files which the job will look for before it runs
outputs = [] # The files which will have to be created in order for the job to be "completed"
options = {'cores': 10, 'memory': "20g", 'walltime': "100:00:00", 'account':"Coryphoideae"} #Slurm commands
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
cd {path_in}
ls *1P.fastq > namelist_temp.txt; sed 's/.........$//' namelist_temp.txt > ../03_hybpiper/namelist.txt; rm namelist_temp.txt
""".format()
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
################################################---- Hybpiper ----######################################################
########################################################################################################################
def hybpiper(species, p1, p2, un, path_out, path_in, done, target_sequence):
"""Hybpiper assemble ."""
inputs = [path_in + species +p1, path_in + species + p2, path_in + species + un] # The files which the job will look for before it runs
outputs = [path_out + species, done] # The files which will have to be created in order for the job to be "completed"
options = {'cores': 10, 'memory': "20g", 'walltime': "100:00:00", 'account':"Coryphoideae"} #Slurm commands
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate hybpiper_env
cd /scratch/$SLURM_JOBID
hybpiper assemble --cpu 10 --readfiles {p1} {p2} --unpaired {un} -b {target_sequence} --prefix {species} --bwa
cp --recursive --update {species} {out}
touch {done}
touch {out}{species}
""".format(species=species, p1 = path_in + species + p1,p2 = path_in + species + p2, un = path_in + species + un , out = path_out, done = done, target_sequence = target_sequence)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
############################################---- Hybpiper stats ----####################################################
########################################################################################################################
def hybpiper_stats(path_out, path_in, done, target_sequence, namelist):
"""Hybpiper stats."""
inputs = [] # The files which the job will look for before it runs
outputs = [done] # The files which will have to be created in order for the job to be "completed"
options = {'cores': 10, 'memory': "20g", 'walltime': "00:30:00", 'account':"Coryphoideae"} #Slurm commands
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate hybpiper_env
hybpiper stats
""".format()
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
#############################################---- Paralogs ----#########################################################
########################################################################################################################
def paralogs(species,path_in, done, no_paralogs, in_done):
"""Find Paralog genes and write them in the file called paralog.txt"""
inputs = [path_in + species, in_done]
outputs = [done]
options = {'cores': 2, 'memory': "10g", 'walltime': "0:30:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate base
if test -f /home/owrisberg/Coryphoideae/work_flow/03_hybpiper/{sp}/genes_with_paralog_warnings.txt; then
echo "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/{sp}/genes_with_paralog_warnings.txt exists"
cd {path_in}
python /home/owrisberg/Coryphoideae/github_code/HybPiper/paralog_investigator.py {sp} 2>> paralog.txt
else
echo "the genes_with_paralog_warnings.txt does not exist and we run the no parallels part"
touch {np}
fi
touch {done}
""".format(sp = species, done = done, path_in = path_in, np = no_paralogs)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
def no_paralogs(species, path_in, done, no_paralogs):
"""Wrapper script to continue pipeline when Hybpiper finds no paralogs"""
inputs = [path_in + species]
outputs = [done]
options = {'cores': 2, 'memory': "10g", 'walltime': "0:05:00", 'account':"Coryphoideae"}
spec = """
touch {done}
touch {np}
""".format(done=done, np=no_paralogs)
returnAnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
# ########################################################################################################################
# #############################################---- Intronerate ----######################################################
# ########################################################################################################################
def intronerate(species, path_in, done):
"""Intronerate the sequencec from hybpiper."""
inputs = [path_in + species, path_in+"done/Hybpiper/"+species]
outputs = [done]
options = {'cores': 4, 'memory': "20g", 'walltime': "16:00:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate base
cd {path_in}
python3 /home/owrisberg/Coryphoideae/github_code/HybPiper/intronerate.py --prefix {sp} &>> intronerate_out.txt
touch {done}
""".format(sp = species, done = done, path_in = path_in)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
# ########################################################################################################################
# #############################################---- Coverage ----#########################################################
# ########################################################################################################################
def coverage(species, path_in, path_out, done,all_bam,all_sorted_bam, all_sorted_bam_bai, bam, cov,fasta,fasta_amb,fasta_ann,fasta_bwt,fasta_pac,fasta_sa,trimmed_fasta,up_bam,dir_in,dir_out):
"""Calculating coverage of sequences."""
inputs = [path_in+species, path_in+"done/Intronerate/"+species]
outputs = [path_out+species+all_bam,
path_out+species+all_sorted_bam,
path_out+species+all_sorted_bam_bai,
path_out+species+bam,
path_out+species+cov,
path_out+species+fasta,
path_out+species+fasta_amb,
path_out+species+fasta_ann,
path_out+species+fasta_bwt,
path_out+species+fasta_pac,
path_out+species+fasta_sa,
path_out+species+trimmed_fasta,
path_out+species+up_bam,done] #ALL the output files
options = {'cores': 4, 'memory': "20g", 'walltime': "08:00:00", 'account':"Coryphoideae"}
spec = """
source /home/owrisberg/miniconda3/etc/profile.d/conda.sh
conda activate base
cd {path_in}
python3 /home/owrisberg/Coryphoideae/github_code/coryphoideae_species_tree/coverage.py {sp} {dir_in} {dir_out}
touch {done}
""".format(sp = species, done = done, path_in = path_in, dir_in = dir_in, dir_out = dir_out)
return AnonymousTarget(inputs=inputs, outputs=outputs, options=options, spec=spec)
########################################################################################################################
##############################################-- Setting up the global variables --#####################################
########################################################################################################################
# Setting up some global variables with relative paths
script_dir = os.path.dirname(getsourcefile(renamer)) + "/"
data_dir = os.path.normpath(os.path.join(script_dir, "../../data/")) +"/"
workflow_dir = os.path.normpath(os.path.join(script_dir, "../../work_flow/")) +"/"
conda_executable_path = os.popen('which conda').read().strip()
conda_bin_dir = os.path.dirname(conda_executable_path)
conda_sh_path = os.path.normpath(os.path.join(conda_bin_dir, "../etc/profile.d/conda.sh"))
########################################################################################################################
######################################################---- RUN ----#####################################################
########################################################################################################################
sp = ["1001","1002","1003","1004","1005","1006","1007","1008","1009","1010","1011","1012","1013","1014","1015","1016","1017","1018","1019","1020","1021","1022","1023","1024","1025","1026","1027","1028","1029","1030","1031","1032","1033","1034","1035","1036","1037","1038","1039","1040","1041","1042","1043","1044","1045","1046","1047","1048","1049","1050","1051","1052","1053","1054","1055","1056","1057","1058","1059","1060","1061","1062","1063","1064","1065","1066","1067","1068","1069","1070","1071","1072","1073","1074","1075","1076","1077","1078","1079","1080","1099","1081","1082","1193","1194","1195","1196","1197","1222","1224","1237","1238","1239","1244","1245","1246","1247","1248","1249","1250","1251","1252","1255","1256","1257","1258","1259","1266","1267","1268","1269","1273","1274","1275","1276","1344","1345","1346","1347","1348","1349","1350","1351","1352","1353","1354","1355","1362","1363","1364","1411","1412","1413","1414","1415","1416","1417","1418","1419","1421","1430","1431","1432","1433","1434","1435","1437","1438","1439","1440","1442","1443","1444","1445","1447","1449","1459","1460","1461","1462","1463","1465","1469","1470","1475","1476","1477","1478","1488","1489","1492","2001","2002","2003","2004","2005","2006","2007","2008","2009","2010","2011","2018","2019","2028","2035","2037","2038","2039","2040","2041","2042","2043","2044","2045","2046","2047","2048","2049","2050","2055","2058","2059","2060","2061","2062","2063","2064","2065","2066","2067","2068","2069","3000","3002","3004","3006","3008","3010","3012","3014","3016","3018","3020","3022","3024","3026","3028","3030","3032","3034","3036","3038","3040","3042","3044","3046","3048","3052","3054","3056","3058","3060","3062","3064","3066","3068","3070","3072","3074","3076","3078","3080","3082","3084","3086","3088","3090","3092","3094","3096","3098","3100","3102","3104","3106","3108","3110","3112","3114","3116","3118","3120","3122","3124","3126","3128","3130","3132","3134","3136","3138","3140","3142","3144","3146","3148","3152","3154","3156","3158","3160","3162","3164","3166","3168","3170","3172","3174","3176","3178","3180","3182","3184","3186","3190","3192","3194","3196","3198","3200","3202","3204","3206","3208","3210","3212","3214","3216","3218","3220","3222","3224","3226","3228","3230","3232","3234","3236","3238","3240","3242","3244","3246","3248","3250","3252","3254","3256","3258","3260","3262","3264","3266","3268","3270","3274","3276","3278","3280","3282","3284","3286","3288","3290","3292","3294","3296","3298","3302","3304","3306","3308","3310","3312","3314","3318","3320","3322","3324","3326","3328","3330","3332","3334","3336","3338","3340","3342","3344","3346","3348","3350","3352","3354","3356","3358","3360","3362","3366","3368","3370","3372","3374","3376","3378","3380","3382","3384","3386","3388","3390","3396","3398","3400","3402","3404","3406","3408","3410","3412","3414","3416","3418","3420","3422","3424","3426","3428","3430","3432","3434","3436","3438","3440","3442","3444","4000","4001","4002","4003","4004","4005","4006","4007","4008","4009","4010","4011","4012","4013","4014","4015","4016","4017","4018","4019","4020","4021","4022","4023","4024","4025","4026","4027","4028","4029","4030","4031","4032","4033","4034","4035","4036","4037","4038","4039","4040","4052","4053","4054","4055","4056","4057","4058","4059","4060","4061","4062","4063","4064","4065","4066","4067","4068","4069","4070","4071","4072","4073","4074","4075","4076","4077","4078","4079","4080","4081","4082","4083","4084","4085","4086","4087","4088","4089","4091","4092","4093","4094","4095","4096","4097","4098","4099","4100","4101","4102","4103"]
#Species removed from pipeline as they had no gene recovery [3050,3188,3272,3300,3316, 3364, 3392, 3394]
# These sp were at the end of SP and I think most of them are duplicates but I dont dare delete them out of fear of missing something.
sp_random_duplicates = ["1436","1254","1441","1257","1258","1196","1443","1444","1445","1099","1436","1254","1441","1257","1258","1196","1443","1444","1445","1099","1259","1098"]
# What is this list of species? why havent I commented this ...
sp_2 = ["1436","1254","1255","1441","1257","1258","1196","1443","1444","1445","1099","1259","1098","4101","4102","4103"]
gwf.target_from_template('renamer', renamer(path_in = "/home/owrisberg/Coryphoideae/work_flow/01_data/",
rename_file = script_dir+"names_1_data_1_rename_seqs.csv",
done = "/home/owrisberg/Coryphoideae/work_flow/01_data/done/renamer/done",
remove_file = script_dir+"names_1_data_2_remove_seqs.csv"))
for i in range(len(sp)):
#### Running fastqc on raw data
gwf.target_from_template('fastqc_raw_'+sp[i], fastqc_raw(species = sp[i],
path_in= "/home/owrisberg/Coryphoideae/work_flow/01_data/",
path_out = "/home/owrisberg/Coryphoideae/work_flow/00_secapr/0_data/",
done = "/home/owrisberg/Coryphoideae/work_flow/00_secapr/done/raw_data/"+sp[i]))
#### Running Trimmomatic
gwf.target_from_template('trimmomatic_'+sp[i], trimmomatic(species = sp[i],
path_in= "/home/owrisberg/Coryphoideae/work_flow/01_data/",
path_out = "/home/owrisberg/Coryphoideae/work_flow/02_trimmed/",
done = "/home/owrisberg/Coryphoideae/work_flow/02_trimmed/done/"+sp[i]))
#### Running fastqc on the trimmed data
gwf.target_from_template('fastqc_trimmed_'+sp[i], fastqc_trimmed(species = sp[i],
path_in= "/home/owrisberg/Coryphoideae/work_flow/02_trimmed/secapr_postrim/",
path_out = "/home/owrisberg/Coryphoideae/work_flow/00_secapr/1_trimmed/",
done = "/home/owrisberg/Coryphoideae/work_flow/00_secapr/done/trimmed_data/"+sp[i]))
#### Running Hybpiper
gwf.target_from_template('Hybpiper_'+sp[i], hybpiper(species = sp[i],
p1 = "_1P.fastq",
p2 = "_2P.fastq",
un = "_UN.fastq",
path_out= "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/",
path_in = "/home/owrisberg/Coryphoideae/work_flow/02_trimmed/",
done = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/Hybpiper/"+sp[i],
target_sequence = os.path.join(workflow_dir,"../target_sequence/PhyloPalms_loci_renamed_794-176_HEYcorrected.fasta")))
#### Paralogs
gwf.target_from_template('Paralogs_'+sp[i], paralogs(species = sp[i],
path_in = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/",
done = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/Paralogs/"+sp[i],
no_paralogs="/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/No_paralogs/"+sp[i],
in_done="/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/Hybpiper/"+sp[i]))
# else:
# gwf.target_from_template('No_Paralogs_'+sp[i], no_paralogs(species = sp[i],
# path_in = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/",
# done = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/Paralogs/"+sp[i],
# no_paralogs="/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/No_paralogs/"+sp[i]))
#### Getting introns
gwf.target_from_template('Intronerate_'+sp[i], intronerate(species= sp[i],
path_in = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/",
done = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/done/Intronerate/"+sp[i]))
#### Coverage
gwf.target_from_template('Coverage_'+sp[i], coverage(species = sp[i],
path_in = "/home/owrisberg/Coryphoideae/work_flow/03_hybpiper/",
all_bam = "_all.bam",
all_sorted_bam ="_all_sorted.bam",
all_sorted_bam_bai="_all_sorted.bam.bai",
bam =".bam",
cov=".cov",
fasta = ".fasta",
fasta_amb = ".fasta.amb",
fasta_ann = ".fasta.ann",
fasta_bwt = ".fasta.bwt",
fasta_pac = ".fasta.pac",
fasta_sa = ".fasta.sa",
trimmed_fasta = "_trimmed.fasta",
up_bam = "_up.bam",
path_out = "/home/owrisberg/Coryphoideae/work_flow/04_coverage/",
done = "/home/owrisberg/Coryphoideae/work_flow/04_coverage/done/Coverage/"+sp[i],
dir_in ="/home/owrisberg/Coryphoideae/work_flow/02_trimmed/", #Folder with raw reads
dir_out ="/home/owrisberg/Coryphoideae/work_flow/04_coverage/")) # folder with coverage