-
Notifications
You must be signed in to change notification settings - Fork 74
/
EDTA_processK.1.pl
executable file
·218 lines (188 loc) · 9.72 KB
/
EDTA_processK.1.pl
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
#!/usr/bin/env perl
use warnings;
use strict;
use FindBin;
use File::Basename;
#####################################################################
##### Perform EDTA basic and advance filtering on TE candidates #####
##### Shujun Ou (shujun.ou.1@gmail.com, 12/28/2023) #####
#####################################################################
## Input:
# $genome.SINE.raw.fa
# $genome.LINE.raw.fa
# $genome.LTR.raw.fa
# $genome.LTR.intact.raw.fa
# $genome.TIR.intact.raw.fa
# $genome.Helitron.intact.raw.fa
## Output:
# $genome.EDTA.fa.stg1
my $usage = "\nPerform EDTA basic and advance filtering for raw TE candidates and generate the stage 1 library
perl EDTA_processF.pl [options]
-genome [File] The genome FASTA
-ltr [File] The raw LTR library FASTA
-ltrint [File] The intact LTR library FASTA
-sine [File] The raw SINE library FASTA
-line [File] The raw LINE library FASTA
-tir [File] The raw TIR library FASTA
-helitron [File] The raw Helitron library FASTA
-mindiff_ltr [float] The minimum fold difference in richness between LTRs and contaminants (default: 1)
-mindiff_tir [float] The minimum fold difference in richness between TIRs and contaminants (default: 1)
-mindiff_hel [float] The minimum fold difference in richness between Helitrons and contaminants (default: 2)
-repeatmasker [path] The directory containing RepeatMasker (default: read from ENV)
-blast [path] The directory containing Blastn (default: read from ENV)
-threads|-t [int] Number of theads to run this script
-help|-h Display this help info
\n";
# user input
my $genome = '';
my $LTRraw = '';
my $LTRintact = '';
my $SINEraw = '';
my $LINEraw = '';
my $TIRraw = '';
my $HELraw = '';
my $err = '';
# minimum richness difference between $TE1 and $TE2 for a sequence to be considered as REAL to $TE1.
# Smaller number is more inclusive during purging, hence higher false positives
my $mindiff_LTR = 1;
my $mindiff_TIR = 1;
my $mindiff_HEL = 2;
my $threads = 4;
my $script_path = $FindBin::Bin;
my $TE_purifier = "$script_path/bin/TE_purifier.pl";
my $rename_TE = "$script_path/bin/rename_TE.pl";
my $cleanup_tandem = "$script_path/bin/cleanup_tandem.pl";
my $cleanup_nested = "$script_path/bin/cleanup_nested.pl";
my $cleanup_proteins = "$script_path/bin/cleanup_proteins.pl";
my $repeatmasker = " ";
my $blast = " ";
# read parameters
my $k=0;
foreach (@ARGV){
$genome = $ARGV[$k+1] if /^-genome$/i and $ARGV[$k+1] !~ /^-/;
$LTRraw = $ARGV[$k+1] if /^-ltr$/i and $ARGV[$k+1] !~ /^-/;
$LTRintact = $ARGV[$k+1] if /^-ltrint$/i and $ARGV[$k+1] !~ /^-/;
$SINEraw = $ARGV[$k+1] if /^-sine$/i and $ARGV[$k+1] !~ /^-/;
$LINEraw = $ARGV[$k+1] if /^-line$/i and $ARGV[$k+1] !~ /^-/;
$TIRraw = $ARGV[$k+1] if /^-tir/i and $ARGV[$k+1] !~ /^-/;
$HELraw = $ARGV[$k+1] if /^-helitron/i and $ARGV[$k+1] !~ /^-/;
$mindiff_LTR = $ARGV[$k+1] if /^-mindiff_ltr/i and $ARGV[$k+1] !~ /^-/;
$mindiff_TIR = $ARGV[$k+1] if /^-mindiff_tir/i and $ARGV[$k+1] !~ /^-/;
$mindiff_HEL = $ARGV[$k+1] if /^-mindiff_hel/i and $ARGV[$k+1] !~ /^-/;
$repeatmasker = $ARGV[$k+1] if /^-repeatmasker/i and $ARGV[$k+1] !~ /^-/;
$blast = $ARGV[$k+1] if /^-blast/i and $ARGV[$k+1] !~ /^-/;
$threads = $ARGV[$k+1] if /^-threads$|^-t$/i and $ARGV[$k+1] !~ /^-/;
die $usage if /^-help$|^-h$/i;
$k++;
}
# check files and dependencies
die "Genome file $genome not exists!\n$usage" unless -s $genome;
die "LTR raw library file $LTRraw not exists!\n$usage" unless -s $LTRraw;
die "Intact LTR file $LTRintact not exists!\n$usage" unless -s $LTRintact;
#die "LINE raw library file $LINEraw not exists!\n$usage" unless -e $LINE; # allow empty file
#die "SINE raw library file $SINEraw not exists!\n$usage" unless -e $SINE; # allow empty file
die "TIR raw library file $TIRraw not exists!\n$usage" unless -s $TIRraw;
die "Helitron raw library file $HELraw not exists!\n$usage" unless -s $HELraw;
die "The script TE_purifier.pl is not found in $TE_purifier!\n" unless -s $TE_purifier;
die "The script rename_TE.pl is not found in $rename_TE!\n" unless -s $rename_TE;
die "The script cleanup_tandem.pl is not found in $cleanup_tandem!\n" unless -s $cleanup_tandem;
die "The script cleanup_nested.pl is not found in $cleanup_nested!\n" unless -s $cleanup_nested;
die "The script cleanup_proteins.pl is not found in $cleanup_proteins!\n" unless -s $cleanup_proteins;
# make a softlink to the genome
my $genome_file = basename($genome);
`ln -s $genome $genome_file` unless -e $genome_file;
$genome = $genome_file;
my $LTR = "$genome.LTR.raw.fa";
my $LTRint = "$genome.LTR.intact.raw.fa";
my $SINE = "$genome.SINE.raw.fa";
my $LINE = "$genome.LINE.raw.fa";
my $TIR = "$genome.TIR.intact.raw.fa";
my $HEL = "$genome.Helitron.intact.raw.fa";
# Make working directories
`mkdir $genome.EDTA.combine` unless -e "$genome.EDTA.combine" && -d "$genome.EDTA.combine";
# enter the combine folder for EDTA processing
chdir "$genome.EDTA.combine";
`cp ../$LTRraw $LTR` unless -s "$LTR";
`cp ../$LTRintact $LTRint` unless -s "$LTRint";
`cp ../$SINEraw $SINE` unless -s "$SINE";
`cp ../$LINEraw $LINE` unless -s "$LINE";
`cp ../$TIRraw $TIR` unless -s "$TIR";
`cp ../$HELraw $HEL` unless -s "$HEL";
##################################
###### define subroutines ######
##################################
# purify $TE2 contaminants in $TE1
# This function better works for redundant libraries
sub Purifier() {
my ($TE1, $TE2, $mindiff) = ($_[0], $_[1], $_[2]);
# mark contaminents with lowercase letters based on relative richness
`perl $TE_purifier -TE1 $TE1 -TE2 $TE2 -t $threads -mindiff $mindiff`;
# remove lowercase sequences
`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 1 -minrm 1 -trf 0 -f $TE1-$TE2.fa > $TE1.HQ`;
}
#################################
###### Advance filtering ######
#################################
## Purge contaminants in redundant libraries
# purify raw LTR (clean LTR library is better than dirty intact LTR for purging LTRs from other TEs)
&Purifier("$LTR", "$TIR", $mindiff_LTR);
&Purifier("$LTR.HQ", "$HEL", $mindiff_LTR);
`mv $LTR.HQ.HQ $LTR.HQ`;
# purify Helitron
&Purifier("$HEL", "$TIR", $mindiff_HEL);
&Purifier("$HEL.HQ", "$LTR", $mindiff_HEL);
#`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 0 -minrm 1 -trf 0 -f $HEL.HQ-$LTR.fa > $HEL.int.cln`; # more relaxed in filtering intact helitrons
#`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 1 -minrm 1 -trf 0 -f $HEL.HQ-$LTR.fa > $HEL.int.cln`; # more stringent
`mv $HEL.HQ.HQ $HEL.cln`;
`cp $HEL.cln $HEL.int.cln`;
# purify TIR
&Purifier("$TIR", "$LTR", $mindiff_TIR);
&Purifier("$TIR.HQ", "$HEL", $mindiff_TIR);
`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 0 -minrm 1 -trf 0 -f $TIR.HQ-$HEL.fa > $TIR.int.cln`; # more relaxed in filtering intact TIRs
`mv $TIR.HQ.HQ $TIR.cln`;
# purify intact LTR from TIRs. Including Helitron is too damaging for now.
&Purifier("$LTRint", "$TIR.cln", 10); # 10 is permissive
`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 0 -minrm 1 -trf 0 -f $LTRint-$TIR.cln.fa > $LTRint.cln`;
#&Purifier("$LTRint.HQ", "$HEL.cln", 10); # 10 is permissive
#`perl $cleanup_tandem -misschar l -Nscreen 1 -nc 50000 -nr 0.8 -minlen 80 -cleanN 1 -cleanT 0 -minrm 1 -trf 0 -f $LTRint.HQ-$HEL.cln.fa > $LTRint.cln`; # more relaxed in filtering intact LTRs
## Purge contaminants in non-redundant libraries
# clean LINEs in LTRs
if (-s "$LINE"){
$err = `${repeatmasker}RepeatMasker -e ncbi -pa $threads -q -no_is -nolow -div 40 -lib $LINE $LTR 2>&1`;
if ($err !~ /done/) {
`ln -s $LTR $LTR.masked` if $err =~ s/^.*(No repetitive sequences were detected.*)\s+$/Warning: No sequences were masked/s;
print STDERR "\n$err\n";
}
`perl $cleanup_tandem -misschar N -nc 50000 -nr 0.9 -minlen 80 -minscore 3000 -trf 0 -cleanN 1 -cleanT 1 -f $LTR.masked > $LTR.cln`;
} else {
`cp $LTR $LTR.cln`;
}
# clean LINEs and LTRs in SINEs
if (-s "$SINE"){
`cat $LTR.cln $LINE > $genome.LINE_LTR.raw.fa`;
$err = `${repeatmasker}RepeatMasker -e ncbi -pa $threads -q -no_is -nolow -div 40 -lib $genome.LINE_LTR.raw.fa $SINE 2>&1`;
if ($err !~ /done/) {
`ln -s $SINE $SINE.masked` if $err =~ s/^.*(No repetitive sequences were detected.*)\s+$/Warning: No sequences were masked/s;
print STDERR "\n$err\n";
}
`perl $cleanup_tandem -misschar N -nc 50000 -nr 0.9 -minlen 80 -minscore 3000 -trf 0 -cleanN 1 -f $SINE.masked > $SINE.cln`;
} else {
`cp $SINE $SINE.cln`;
}
## clean LTRs and nonLTRs in TIRs and Helitrons
`cat $TIR.cln $HEL.cln | perl -nle 's/>/\\n>/g unless /^>/; print \$_' > $genome.TIR.Helitron.fa.stg1.raw`;
`cat $LTR.HQ $SINE.cln $LINE > $genome.LTR.SINE.LINE.fa`;
$err = `${repeatmasker}RepeatMasker -e ncbi -pa $threads -q -no_is -nolow -div 40 -lib $genome.LTR.SINE.LINE.fa $genome.TIR.Helitron.fa.stg1.raw 2>&1`;
if ($err !~ /done/) {
`ln -s $genome.TIR.Helitron.fa.stg1.raw $genome.TIR.Helitron.fa.stg1.raw.masked` if $err =~ s/^.*(No repetitive sequences were detected.*)\s+$/Warning: No sequences were masked/s;
print STDERR "\n$err\n";
}
`perl $cleanup_tandem -misschar N -nc 50000 -nr 0.9 -minlen 80 -minscore 3000 -trf 0 -cleanN 1 -cleanT 1 -f $genome.TIR.Helitron.fa.stg1.raw.masked > $genome.TIR.Helitron.fa.stg1.raw.cln`;
## cluster TIRs and Helitrons and make stg1 raw library
`perl $cleanup_nested -in $genome.TIR.Helitron.fa.stg1.raw.cln -threads $threads -minlen 80 -cov 0.95 -blastplus $blast`;
`cat $LTR $LINE $SINE.cln $genome.TIR.Helitron.fa.stg1.raw.cln.cln > $genome.EDTA.fa.stg1`;
## generate clean intact TEs
`cat $LTRint.cln $LINE $SINE.cln $TIR.int.cln $HEL.int.cln > $genome.EDTA.intact.fa.cln`;
## clean up the folder
`rm *.ndb *.not *.ntf *.nto *.cat.gz *.cat *.masked *.ori.out *.nhr *.nin *.nsq 2>/dev/null`;
chdir '..';