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import QCumber
import datetime
import numpy as np
import getpass
import csv
from helper import *
import re
from os.path import basename, splitext, join, exists, dirname, isdir
from os import uname, listdir, remove
import configparser
import shutil
from collections import OrderedDict
import base64
default_parameter = configparser.ConfigParser()
default_parameter.read(join(dirname(__file__), "parameter.txt"))
def get_tool_path(name, section="PATH"):
config = configparser.ConfigParser()
config.read(join(dirname(__file__), "config.txt"))
path_dict = config[section]
return path_dict[name]
class Pipeline:
'''Get all tool versions '''
def __init__(self):
self.name = "Quality Control"
self.version = QCumber.__version__
self.user = getpass.getuser()
self.python_version = toLatex(sys.version)
self.fastqc_version = toLatex(subprocess.check_output(join(get_tool_path("fastqc"), "fastqc") + " --version", shell = True, stderr=subprocess.PIPE).decode("utf-8"))
try:
self.trimmo_version = subprocess.check_output(join(get_tool_path("trimmomatic"), "TrimmomaticSE") + " -version", shell=True, stderr=subprocess.PIPE).decode("utf-8")
except:
try:
from debpackageinfo import get_upstream_version
self.trimmo_version = get_upstream_version("trimmomatic")
#self.trimmo_version = re.search("Trimmomatic-(?P<version>\d.\d+)",get_tool_path("trimmomatic")).group("version")
except:
self.trimmo_version = "< 0.32"
bowtie_v = subprocess.check_output(join(get_tool_path("bowtie2"), "bowtie2") + " --version", shell=True).decode("utf-8")
self.bowtie_version = re.match(".*\)", toLatex(bowtie_v, True)).group()
self.kraken_version =toLatex(subprocess.check_output( join(get_tool_path('kraken'), "kraken") + " --version", shell = True).decode("utf-8"))
system = uname()
self.system = toLatex(system.sysname)
self.server = toLatex(system.nodename)
self.release = toLatex(system.release)
self.operating_version = toLatex(system.version)
self.machine = toLatex(system.machine)
class Sample(object):
"""Sample with reference to ReadSets, MappingRes, KrakenResr"""
def __init__(self, name, path, reference, threads, techn):
global mainResultsPath
global num_threads
global technology
num_threads = threads
mainResultsPath = path
technology = techn
self.start = datetime.datetime.strftime(datetime.datetime.now(),"%d.%m.%Y, %H:%M:%S")
self.stop = None
self.name = name
self.technology = techn
self.threads = threads
self.phred = "phred33"
self.readSets = []
self.mainResultsPath = path
self.mappingRes = None
self.krakenRes = None
self.reference = reference
def get_all_qc_plots(self):
qc_dict = OrderedDict([("setname", self.name),("raw",[]), ("trimmed",[])])
for rs in self.readSets:
qc_dict["raw"].append(rs.r1.qcRes.plots)
qc_dict["trimmed"].append(rs.trimRes.readset.r1.qcRes.plots)
if (rs.r2 is not None):
qc_dict["raw"].append(rs.r2.qcRes.plots)
qc_dict["trimmed"].append(rs.trimRes.readset.r2.qcRes.plots)
return qc_dict
def total_reads(self):
total = 0
for rs in self.readSets:
total += rs.numberOfReads
return total
def pTrimmed(self):
perc = []
for rs in self.readSets:
if rs.trimRes:
perc.append(rs.trimRes.pTrimmed)
if len(perc) == 0 :
return 0
return round(np.mean(perc), 2)
def nTrimmed(self):
total = 0
for rs in self.readSets:
if rs.trimRes:
total += rs.trimRes.nTrimmed
return total
def add_readSet(self, readSet):
self.readSets.append(readSet)
return self
def run_Kraken(self, db, trimmed):
r1, r2 = self.get_all_reads(trimmed)
self.krakenRes = KrakenResult(r1,r2,db, self.name)
return self
def run_Bowtie2(self, bowtie_index, save_mapping, trimmed ):
r1, r2 = self.get_all_reads(trimmed)
self.mappingRes = MappingResult(self.reference, r1, r2, bowtie_index)
self.mappingRes = self.mappingRes.run_bowtie(save_mapping)
return self.mappingRes
def get_all_reads(self, trimmed = True):
if trimmed:
r1 = " ".join([x.trimRes.readset.r1.filename for x in self.readSets])#join_reads([x.trimRes.readset.r1.filename for x in self.readSets], self.mainResultsPath, self.name + "_R1")
r2 = " ".join([x.trimRes.readset.r2.filename for x in self.readSets if x.trimRes.readset.r2 is not None])#join_reads([x.trimRes.readset.r2.filename for x in self.readSets if x.trimRes.readset.r2 is not None], self.mainResultsPath, self.name + "_R2")
else:
r1 = " ".join([x.r1.filename for x in self.readSets])#join_reads([x.r1.filename for x in self.readSets], self.mainResultsPath, self.name + "_R1")
r2 = " ".join([x.r2.filename for x in self.readSets if x.r2 is not None]) #join_reads([x.r2.filename for x in self.readSets if x.r2 is not None], self.mainResultsPath, self.name + "_R2") #readnames = {'r1' : r1}
return r1,r2
def get_mean_trimRes(self):
try:
return round(np.mean([x.trimRes.shorter_fragments_percentage for x in self.readSets]),2)
except:
return None
class ReadSet:
''' Readset contains of r1 and r2'''
def __init__(self, r1 ,r2=None):
self.r2 = None
if type(r1) is str:
self.r1 = FastQFile(r1) # FastQFile
if r2 is not None:
self.r2 = FastQFile(r2) # FastQFile
else:
self.r1 = r1
if r2 is not None:
self.r2 = r2
self.numberOfReads = 0
self.numberofTrimmedReads = 0
self.trimRes = None
self.paired = True
if len( basename(self.r1.filename).split("_")[:-3]) > 4:
self.setname = "_".join(basename(self.r1.filename).split("_")[:-3])
else:
self.setname = basename(self.r1.filename)
if(r2 is None):
self.paired = False
def run_FastQC(self, output):
self.r1 = self.r1.run_FastQC(output)
self.numberOfReads += self.r1.qcRes.total_reads
if(self.r2 is not None):
self.r2 = self.r2.run_FastQC(output)
self.numberOfReads += self.r1.qcRes.total_reads
return self
def run_Trimmomatic(self, adapter, palindrome, minlen, trimOption, betterTrimming,gz):
self.trimRes = TrimResult(self, adapter, palindrome, minlen, trimOption, betterTrimming,gz )
return self
def get_all_qc(self):
qc_results = [ (self.r1.qcRes, self.trimRes.readset.r1.qcRes) ]
if (self.r2 is not None):
qc_results.append((self.r2.qcRes, self.trimRes.readset.r2.qcRes) )
return qc_results
def get_all_qc_dict(self):
qc_dict = OrderedDict({"raw": [], "trimmed": []})
qc_dict["raw"] = [[x.__dict__ for x in self.r1.qcRes.results_for_report()]]
if self.trimRes:
qc_dict["trimmed"] = [[x.__dict__ for x in self.trimRes.readset.r1.qcRes.results_for_report()]]
if (self.r2 is not None):
qc_dict["raw"].append([x.__dict__ for x in self.r2.qcRes.results_for_report()])
if self.trimRes:
qc_dict["trimmed"].append([x.__dict__ for x in self.trimRes.readset.r2.qcRes.results_for_report()])
return qc_dict
class FastQFile:
def __init__(self, absFilename, concat_files = None):
self.filename = absFilename
self.qcRes = None
#self.log = ""
self.phred="phred33"
self.concat_files = None
def run_FastQC(self, outputDir):
process = subprocess.Popen([join(get_tool_path('fastqc'), "fastqc") , self.filename , "-o" , outputDir, "-t", str(num_threads), "--extract"], stdout = subprocess.PIPE, stderr = subprocess.PIPE)
for line in iter(process.stderr.readline, b''):
line = line.decode("utf-8")
line = line.replace("\n", "")
print(line, end="\r")
for line in iter(process.stdout.readline, b''):
line = line.decode("utf-8")
if line.startswith("Approx"):
print(line, end="\r")
else:
print(line, end="\r")
pattern = re.match("Quality encoding detected as (?P<phred>\w+\d{2})", line)
if pattern:
self.phred = pattern.group("phred")
process.communicate()
self.qcRes = QCResult(join(outputDir, getFilenameWithoutExtension(basename(self.filename)) + "_fastqc"), join(mainResultsPath, "Report", "src"))
return self
def get_filename(self):
return toLatex(basename(self.filename))
###########
## Result classes
###########
class QCResult:
def __init__(self, resultsPath, summary_output):
self.path = resultsPath
self.results = self.get_results()
#write summary csv for boxplots and assign total number of reads
self.total_reads = write_fastqc_summary(resultsPath, summary_output)
def get_results(self):
summary_list = []
no_plot=["Basic Statistics", "Overrepresented sequences"]
with open(join(self.path, "summary.txt"), "r") as summary:
reader = csv.reader(summary, delimiter = "\t")
for row in reader:
if (row[1] not in no_plot):
try:
plot = Plot(row[0], row[1], self.path)
if exists(plot.file):
summary_list.append(plot)
else:
print("No plot file ", plot.file, row[1])
summary_list.append(Plot("NotExisting", row[1], "NotExisting"))
except:
print(row[1], "does not exist")
summary_list.append(Plot("NotExisting", row[1], "NotExisting"))
return summary_list
def results_to_base64(self):
converted_plot = []
for plot in self.results:
if not plot.file.startswith("NotExisting"):
with open(plot.file, "rb") as imgfile:
imgstring = base64.b64encode(imgfile.read())
plot.file ='data:image/png;base64,' + imgstring.decode("utf-8")
converted_plot.append(plot)
return converted_plot
def results_for_report(self):
converted_plot = []
for plot in self.results:
if not plot.file.startswith("NotExisting"):
plot.file =plot.file.replace(mainResultsPath, "..")
converted_plot.append(plot)
return converted_plot
class Plot:
def __init__(self, value, name, resultsPath):
self.color = self.get_color(value)
self.name = name
self.file = self.name_to_file(resultsPath)
def get_color(self, value):
if(value == "PASS"):
return "green"
elif(value =="FAIL"):
return "red"
#elif (value =="WARN"):
# return "orange"
else:
return "orange"#"grey"
def name_to_file(self,resultsPath):
if self.name in default_parameter["FastQC"].keys():
return join(resultsPath, "Images", default_parameter["FastQC"][self.name])
else:
return ""
class TrimParameters:
illuminaClip_seedMismatch = str(default_parameter["Trimmomatic"]["illuminaClip_seedMismatch"]) #"2" #specifies the maximum mismatch count which will still allow a full match to be performed
illuminaClip_simpleClip = str(default_parameter["Trimmomatic"]["illuminaClip_simpleClip"]) # "10" #specifies how accurate the match between any adapter etc. sequence must be against a read
leading = str(default_parameter["Trimmomatic"]["leading"]) #"3" # remove leading low quality below 3
trailing = str(default_parameter["Trimmomatic"]["trailing"]) #"3" # remove trailing quality below 3
adapter_path = get_tool_path('adapter')
def __init__(self):
#self.palindrome = palindrome
self.extraTrimOptions = ""
def make_callstring(self, adapter,palindrome, minlen, trimOption, betterTrimmingOption ):
call = []
if technology =="Illumina":
call.append("ILLUMINACLIP:" + join(self.adapter_path, adapter+ ".fa") + ":" + self.illuminaClip_seedMismatch + ":" + str(palindrome) + ":" + self.illuminaClip_simpleClip)
call.append(trimOption)
call.append(betterTrimmingOption)
call.append("MINLEN:" + str(minlen))
return " ".join(call)
class TrimResult:
def __init__(self, readset, adapter, palindrome, minlen, trimOption, betterTrimming,gz):
self.parameters = TrimParameters()
self.readset = readset
self.r1_paired = None
self.r2_paired = None
self.r1_unpaired = None
self.r2_unpaired = None
self.logs = ""
#Results
self.total=0
self.shorter_fragments_percentage = 0
self.input_number = 0
self.nTrimmed = 0
self.pTrimmed = 0
self.adapter = adapter
self.run( palindrome, minlen, trimOption, betterTrimming, gz)
def make_callstring(self, readset, palindrome, minlen, trimOption, save = False, betterTrimmingOption ="", gz=True):
if gz:
extension= ".gz"
else:
extension =""
if readset.paired:
if save:
self.r1_paired =join(mainResultsPath, "Trimmed", "r1_P_" + getFilenameWithoutExtension(readset.r1.filename, getBase = True) + ".fastq" + extension)
self.r1_unpaired = join(mainResultsPath, "Trimmed", "r1_UP_" + getFilenameWithoutExtension(readset.r1.filename, getBase = True)+ ".fastq"+ extension)
self.r2_paired =join(mainResultsPath, "Trimmed", "r2_P_" + getFilenameWithoutExtension(readset.r2.filename, getBase = True)+ ".fastq"+ extension)
self.r2_unpaired =join(mainResultsPath, "Trimmed", "r2_UP_" + getFilenameWithoutExtension(readset.r2.filename, getBase = True)+ ".fastq"+ extension)
output = " ".join([self.r1_paired, self.r1_unpaired, self.r2_paired, self.r2_unpaired])
else:
output = "/dev/null /dev/null /dev/null /dev/null"
callProcess = [join(get_tool_path("trimmomatic"), "TrimmomaticPE "),
"-threads", str(num_threads),
"-" + readset.r1.phred,
readset.r1.filename, # input 1
readset.r2.filename, # input 2
output,
self.parameters.make_callstring(self.adapter,palindrome, minlen, trimOption, betterTrimmingOption)
]
else:
callProcess = [join(get_tool_path("trimmomatic"),"TrimmomaticSE "),
"-threads", str(num_threads),
"-" + readset.r1.phred,
readset.r1.filename, # input 1
join(mainResultsPath, "Trimmed", getFilenameWithoutExtension(readset.r1.filename, getBase = True)+ ".fastq"),#output
self.parameters.make_callstring(self.adapter, palindrome, minlen, trimOption, betterTrimmingOption)
]
self.r1_unpaired = join(mainResultsPath, "Trimmed", getFilenameWithoutExtension(readset.r1.filename, getBase = True)+ ".fastq"+ extension)
return callProcess
def check_quality(self,readset, trim_perc):
path = join(mainResultsPath, "Trimmed", "temp")
if readset.paired:
try:
r1_name = join_reads([self.r1_paired, self.r1_unpaired], path, basename(readset.r1.filename))
print("Run FastQC to optimize trimming.")
process = subprocess.Popen( [join(get_tool_path('fastqc'), "fastqc"), r1_name,"--nogroup", "--extract", "-o", path,"--threads", str(num_threads)], stdout=subprocess.PIPE, stderr=subprocess.PIPE)
process.communicate()
trim_bool, headcrop, crop = optimize_trimming(join(path, getFilenameWithoutExtension(basename(r1_name)) + "_fastqc"), trim_perc)
#if r1 was ok, go on and check r2
print("Check 'per sequence base content' of R2.")
r2_name = join_reads([self.r2_paired, self.r2_unpaired], path, basename(readset.r2.filename))
process = subprocess.Popen(
[join(get_tool_path('fastqc'), "fastqc"), r2_name,"--nogroup", "--extract", "-o",path, "--threads", str(num_threads)],
stdout=subprocess.PIPE, stderr=subprocess.PIPE)
process.communicate()
r2_trim_bool, r2_headcrop, r2_crop = optimize_trimming(join(path, getFilenameWithoutExtension(basename(r2_name)) + "_fastqc"), trim_perc)
return (trim_bool or r2_trim_bool), max(headcrop, r2_headcrop), min(crop, r2_crop)
finally:
shutil.rmtree(path)
else:
process = subprocess.Popen([join(get_tool_path('fastqc'), "fastqc"), self.r1_unpaired,"--nogroup", "--extract", "-o", path, "--threads", str(num_threads)],
stdout=subprocess.PIPE, stderr=subprocess.PIPE)
for line in iter(process.stdout.readline, b''):
print(line, end='\r')
process.communicate()
trim_bool, headcrop, crop = optimize_trimming(join(path,getFilenameWithoutExtension(basename(self.r1_unpaired)) + "_fastqc"), trim_perc)
return trim_bool, headcrop, crop
def run(self, palindrome, minlen, trimOption, betterTrimming, gz):
# Report Trimmomatic Output
# Run palindrome parameter which should be kept at last
survived = {'30':0, '1000':0}
call = self.make_callstring( self.readset, palindrome, minlen, trimOption, save=True, gz=gz)
process = subprocess.Popen(" ".join(call), stdout=subprocess.PIPE, stderr=subprocess.PIPE, shell=True)
survived1, total, nTrimmedReads, pTrimmedReads, log = trimmomatic_report(process,call[0], True)
process.communicate()
### Optimize trimming
if betterTrimming !="":
print("Improve trimming parameter")
makedirs(join(mainResultsPath, "Trimmed","temp"), exist_ok=True)
trim_bool, final_headcrop, final_crop = self.check_quality(self.readset, betterTrimming)
if trim_bool:
self.parameters.extraTrimOptions = "HEADCROP:"+str(final_headcrop) + " CROP:"+str(final_crop)
call = self.make_callstring( self.readset, palindrome, minlen, trimOption, save=True, betterTrimmingOption = self.parameters.extraTrimOptions, gz=gz)
process = subprocess.Popen(" ".join(call), stdout=subprocess.PIPE, stderr=subprocess.PIPE, shell=True)
survived1, total, nTrimmedReads, pTrimmedReads, log = trimmomatic_report(process, call[0], True)
process.communicate()
if survived1==0:
sys.exit("No reads remained after trimming. Check fastqc results before trimming in QCResults/Fastqc. Please rerun without -trimBetter or check -trimOptions and minlen again.")
self.total = total
self.nTrimmed = nTrimmedReads
self.pTrimmed = pTrimmedReads
self.logs += log
############################
survived[str(palindrome)] = survived1
if self.readset.r2 is not None:
if palindrome==30:
palindrome=1000
else:
palindrome=30
process = subprocess.Popen(" ".join(self.make_callstring( self.readset, palindrome, minlen, trimOption, save=False, betterTrimmingOption = self.parameters.extraTrimOptions, gz=gz) ),
stdout=subprocess.PIPE,
stderr=subprocess.PIPE, shell=True)
survived2, total,nTrimmedReads, pTrimmedReads, temp = trimmomatic_report(process, call[0],False)
process.communicate()
survived[str(palindrome)] = survived2
self.shorter_fragments_percentage = round(survived["1000"] - survived["30"], 2)
return self
def run_FastQC(self, tempdir):
if self.readset.paired:
#tempdir = tempfile.mkdtemp()
r1_name = join_reads([self.r1_paired, self.r1_unpaired], tempdir, basename(self.readset.r1.filename))
r2_name = join_reads([self.r2_paired, self.r2_unpaired], tempdir, basename(self.readset.r2.filename))
try:
self.readset = ReadSet( r1_name, r2_name)
self.readset.run_FastQC(join(mainResultsPath, "Trimmed/FastQC"))
except:
print(sys.exc_info())
sys.exit()
finally:
#remove(r1_name)
#remove(r2_name)
self.readset.r1.filename = " ".join([self.r1_paired, self.r1_unpaired])
self.readset.r2.filename = " ".join([self.r2_paired, self.r2_unpaired])
else:
self.readset = ReadSet( self.r1_unpaired)
self.readset.run_FastQC(join(mainResultsPath, "Trimmed"))
return self
def print_param(self, palindrome, minlen, trimOption):
return self.parameters.make_callstring(self.adapter, palindrome, minlen, trimOption, self.parameters.extraTrimOptions)
class MappingParameter:
def __init__(self, mapping):
pass
class MappingResult:
def __init__(self, reference, r1_name, r2_name, bowtie_index):
self.numberOfAlignedReads = 0
self.percentAlignedReads= 0
self.index = self.build_index(reference, bowtie_index)
self.log = ""
self.r1_name = r1_name
self.r2_name = r2_name
def build_index(self, reference, index):
index_name = join(index, getFilenameWithoutExtension(basename(reference)) +"_bt2") # reference name "bowtie2 build index_name"
if not isdir(index):
return index
if not exists(index_name + ".1.bt2"):
process = subprocess.Popen([join(get_tool_path("bowtie2"),"bowtie2-build"),reference, index_name ], stderr=subprocess.PIPE, stdout = subprocess.PIPE)
for line in iter(process.stderr.readline, b''):
line = line.decode("utf-8")
print(line)
process.communicate()
print("Bowtie2-build. Done.")
return index_name
def run_bowtie(self, save_mapping):
print("Run Bowtie2")
call = [join(get_tool_path("bowtie2"), "bowtie2"), "-x", self.index, "--no-unal --threads ", str(num_threads)]
#if self.r2_name is not None:
# call.extend(["-1", re.sub(r"(?<!\\)\s", ",", self.r1_name)])
# call.extend(['-2',re.sub(r"(?<!\\)\s",",",self.r2_name)])
#else:
# call.extend(["-U", re.sub(r"(?<!\\)\s", ",", self.r1_name)])
call.extend(["-U", re.sub(r"(?<!\\)\s", ",", self.r1_name + " " + self.r2_name)])
call.extend(["-S", save_mapping, "2>&1"])
#print("Call ", " ".join(call))
process = subprocess.Popen(" ".join(call), stderr=subprocess.PIPE, stdout= subprocess.PIPE, shell=True)
for line in iter(process.stdout.readline, b''):
line = line.decode("utf-8")
print(line)
self.log += toLatex(line) + "\n"
pattern1 = re.match(".* (?P<aligned_exact>\d+) \((?P<percent_aligned_exact>\d+.\d+)%\) aligned exactly 1 time", line)
pattern2 = re.match(".* (?P<aligned_more_than_one>\d+) \((?P<percent_aligned_more_than_one>\d+.\d+)%\) aligned >1 times", line)
if pattern1:
self.numberOfAlignedReads += int(pattern1.group("aligned_exact"))
self.percentAlignedReads += float(pattern1.group("percent_aligned_exact").replace(",","."))
if pattern2:
self.numberOfAlignedReads += int(pattern2.group("aligned_more_than_one"))
self.percentAlignedReads += float(pattern2.group("percent_aligned_more_than_one"))
self.percentAlignedReads = round(self.percentAlignedReads,2)
for line in iter(process.stderr.readline, b''):
line = line.decode("utf-8")
self.log += "\\textcolor{red}{" + toLatex(line) + "}\n"
print(line)
process.communicate()
print("Bowtie2 done.")
return self
class KrakenResult:
def __init__(self, r1,r2, db, name):
self.name = name
self.report_name = None
self.report = ""
self.pClassified = 0
self.nClassified = 0
self.log = ""
self.run_kraken(r1,r2, db)
def make_callstring(self, r1,r2, db):
#input_type = ""
#if fileext.endswith("")
call = [join(get_tool_path('kraken') , "kraken") , "--db", db, r1]
if r2 is not None:
call.append( r2)# + " --paired")
call.append("--preload")
call.append("--threads " + str(num_threads))
self.report_name = join(mainResultsPath, self.name + ".krakenreport")
if exists(self.report_name):
i = 2
new_name = join(mainResultsPath, self.name + "_" +str(i) + ".krakenreport")
while exists(new_name):
i += 1
new_name = join(mainResultsPath, self.name + "_" + str(i) + ".krakenreport")
self.report_name=new_name
call.append("--output " + self.report_name)
return " ".join(call)
def run_kraken(self, r1,r2, db):
process = subprocess.Popen(self.make_callstring(r1,r2, db), shell=True, stderr=subprocess.PIPE, stdout=subprocess.PIPE)
self.log += "\\textcolor{gray}{Using Parameter " + " ".join(["\path{"+ x + "}" for x in self.make_callstring(r1,r2, db).split(" ")]) + "}\\\\"
for line in iter(process.stderr.readline, b''):
line = line.decode("utf-8")
print(line)
if not (line.startswith("Loading database") or re.search("Processed \d+", line)):
self.log += toLatex(line) +"\n"
pattern = re.match("\s+(?P<nClassified>\d+) sequences classified \((?P<pClassified>\d+.\d+)%\)", line)
if pattern:
self.nClassified = pattern.group("nClassified")
self.pClassified = pattern.group("pClassified")
for line in iter(process.stdout.readline, b''):
line = line.decode("utf-8")
print(line)
if not ( re.search("Processed \d+", line) or line.startswith("Loading database") ):
self.output += line + "\n"
process.communicate()
print("Convert Kraken-Report..")
self.convert_report(db)
return self
def convert_report(self, db):
process = subprocess.check_output([join(get_tool_path('kraken') ,'kraken-report') , "--db", db, self.report_name])
self.report = process.decode("utf-8")
return self
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