Possible precedence issue with control flow operator at epiTEome.pl line 482.

[CS791]

root@sc-Latitude-E6230:/usr/bin/epiTEome# idxEpiTEome.pl -l 85 -gff tair10TEs.gff3 -t teid.lst -fasta Chr2.fasta
idxEpiTEome.pl: command not found
(base) root@sc-Latitude-E6230:/usr/bin/epiTEome# perl idxEpiTEome.pl -l 85 -gff test/tair10TEs.gff3 -t test/teid.lst -fasta test/Chr2.fasta
INFO idxEpiTEome.pl: All programs have been found succesfully.
INFO idxEpiTEome.pl: Run Module: Tue Jun 4 14:32:52 2019 readGffFile !
INFO idxEpiTEome.pl: Reading GFF file ... done !
INFO idxEpiTEome.pl: Run Module: Tue Jun 4 14:32:54 2019 maskFastaIndex !
/root/anaconda3/bin/maskFastaFromBed -fi test/Chr2.fasta -bed /usr/bin/epiTEome/prepRefSeq.bed -fo test/Chr2.epiTEome.masked.fasta
/root/anaconda3/bin/segemehl.x --silent -x test/Chr2.epiTEome.masked.fasta.ctidx -y test/Chr2.epiTEome.masked.fasta.gaidx -d test/Chr2.epiTEome.masked.fasta -F 1 2> log
All files have been created succesfully !

root@sc-Latitude-E6230:/usr/bin/epiTEome# perl epiTEome.pl -gff test/tair10TEs.gff3 -ref test/Chr2.epiTEome.masked.fasta -un test/unmapped.fastq -t test/teid.lst
Possible precedence issue with control flow operator at epiTEome.pl line 482.
INFO epiTEome.pl Tue Jun 4 14:33:55 2019 Start program!
DIE epiTEome.pl: Could not find segemehl index file .ctidx

HELP: epiTEome.pl

AUTHOR: Josquin DARON, Slotkin Lab, Ohio State University
VERSION: 1.0 -- 2017-02-01

PURPOSE: Identify non-reference TE insertion sites and their methylation level.

USAGE: epiTEome.pl —l [max read length] -gff -t —ref -un

    <gff>     TE annotation should be given in gff3 format.
              For TE annotated features, column 9 should have following list of tags:
              ID (teid), sF (superfamily name), fam (family name).
              For LTR annotated features, they should be referred as LTR5 or LTR3 in column 3,
              column 9 should have tags Parent (teid).
        
    <target>  list of TEid of interest

    <fasta>   FASTA formated (.fa, .fna or fasta) genome file.

    <fastq>   FASTQ file of reads that failed to map the reference genome (unmapped reads)

OPTIONS
  EpiTEome Specific Options:
    -chop [integer] : read ends length of chopped (defaut 25,30,40).
                      Use of several length will improve epiTEome sensitivity. 
    -b    [integer] : number of TE per batch (defaut 5000).
    -w    [integer] : window size for methylation metaplot analysis.

  Alignment Options:
    -E    [integer] : segemehl max evalue (default:5)
    -p    [integer] : number of threads use in segemehl (defaut 1).
                      All other portions are single-threaded.

OUTPUT
  epiTEome output 4 different files such as .newInsertionSite.tab, .newInsertionSite.sam, .met.meta.tab and .met.row.ta

any help much appreciated.

Thank you!
Regards

CS791

[CS791]

Dear Memebers,

I am still facing the problem mentioned above. "idxEpiTEome.pl " generates only master.fasta files but not .ctidx and gtidx. Its annoying as
However, it shows that it generated all file successfully.

perl idxEpiTEome.pl -gff tair10TEs.gff3 -t teid.lst -fasta TAIR10_chr_all.fas -l 85
INFO idxEpiTEome.pl: All programs have been found succesfully.
INFO idxEpiTEome.pl: Run Module: Wed Jun 26 10:36:28 2019 readGffFile !
INFO idxEpiTEome.pl: Reading GFF file ... done !
INFO idxEpiTEome.pl: Run Module: Wed Jun 26 10:36:30 2019 maskFastaIndex !
/usr/local/bin/maskFastaFromBed -fi TAIR10_chr_all.fas -bed /home/sc/epiTEome/prepRefSeq.bed -fo TAIR10_chr_all.epiTEome.masked.fasta
/home/sc/anaconda2/bin/segemehl.x --silent -x TAIR10_chr_all.epiTEome.masked.fasta.ctidx -y TAIR10_chr_all.epiTEome.masked.fasta.gaidx -d TAIR10_chr_all.epiTEome.masked.fasta -F 1 2> log
All files have been created succesfully !

I may be missing any minor step. But I am not able to figure out.
waiting for any help.

Thank You!
Regards
CS791

[jdaron]

Dear CS791,

I am sorry your facing this issue. In which version of segemehl are you working with ?
EpiTEome has been developed for the version : 0.2.0-418 (2015-01-05 05:17:35 -0500 (Mon, 05 Jan 2015))
I know that recently segemehl released a new version of it tools, this may be the problem here.

Regards,
Josquin

[CS791]

Thank you! Josquin for prompt reply. Yes! I was using new version of segemehl. Now I will try with version : 0.2.0-418 and see.
Hope it works.

Regards
CS791

[CS791]

Hi! Josquin,

Segemehl_0.2.0 is working fine now, but I stuck on another step while running the test run.

Here is the command line and issue:

sc@sc-HP-EliteBook-820-G2:~/epiTEome$ perl epiTEome.pl -gff test/tair10TEs.gff3 -ref test/Chr2.epiTEome.masked.fasta -un test/unmapped.fastq -t test/teid.lst
Possible precedence issue with control flow operator at epiTEome.pl line 482.
INFO epiTEome.pl Wed Jun 26 14:40:03 2019 Start program!
INFO epiTEome.pl Wed Jun 26 14:40:03 2019 Run Module: readGffFile!

INFO epiTEome.pl Wed Jun 26 14:40:05 2019 STEP 1: read ends mapping.
INFO epiTEome.pl Wed Jun 26 14:40:05 2019 Run Module: splitFastq!
INFO epiTEome.pl Wed Jun 26 14:40:05 2019 Run Module: Segemehl mapping !
/usr/bin/segemehl.x --silent -t 1 -D 1 -E 5 -i test/Chr2.epiTEome.masked.fasta.ctidx -j test/Chr2.epiTEome.masked.fasta.gaidx -d test/Chr2.epiTEome.masked.fasta -q test/unmapped.pe.fastq -o test/unmapped.step1.sam -F 1 2>log
/usr/bin/samtools view -b test/unmapped.step1.sam | /usr/bin/samtools sort - -o test/unmapped.step1.sort.bam
/usr/bin/samtools index test/unmapped.step1.sort.bam

INFO epiTEome.pl Wed Jun 26 14:40:06 2019 STEP 2: split-reads mapping.

INFO epiTEome.pl Wed Jun 26 14:40:06 2019 BATCH #1.
INFO epiTEome.pl Wed Jun 26 14:40:06 2019 Run Module: selectPairedEndReads !
AT2TE41090 284
INFO epiTEome.pl Wed Jun 26 14:40:06 2019 Run Module: pairedEndGrab !
/home/sc/.local/bin/bamutils filter test/unmapped.step1.sort.bam test/unmapped.matePairedEnd.bam -whitelist test/unmapped.fishReads.list
/home/sc/.local/bin/bamutils: 27: .: Can't open /home/sc/.local/bin/..//venv/bin/activate
/usr/bin/samtools index test/unmapped.matePairedEnd.bam
[E::hts_open_format] Failed to open file test/unmapped.matePairedEnd.bam
samtools index: failed to open "test/unmapped.matePairedEnd.bam": No such file or directory
test/unmapped.matePairedEnd.bam does not exist at epiTEome.pl line 810.

Please suggest something to troubleshoot this.

Thank you!
Regards

CS791

[jdaron]

I think the problem come from the step that launch bamutils filter.

/home/sc/.local/bin/bamutils filter test/unmapped.step1.sort.bam test/unmapped.matePairedEnd.bam -whitelist test/unmapped.fishReads.list

May be you could run the following command line, with the same option and see if it works. I think it's also a problem of version.

bamutils filter inputBam outputBam -whitelist listOfReadsId

Good luck
Josquin

[CS791]

Many thanks Josquin for your help.
I sorted out the problems with re-installing ngsutils and it works fine with test dataset. However, when I applied it for my own data it shows some error in unmapped.fastq files. I know its quality problem with my unmapped read fastq files, but still if you can give any suggestions will be helpful. My original raw data was paired end and I used the following command line for Bismark mapping:
bismark_v0.21.0/bismark ~/Bismark/Genome -bowtie2 --ambiguous --non_directional -unmapped -R 10 score_min L,0,0.6 -N 1 -1 Epiril_22C_R1_L1/Epiril368_22_C_R1_L001_R1_val_1.fq.gz -2 Epiril_22C_R1_L1/Epiril368_22_C_R1_L001_R2_val_1.fq.gz -o New_Output/Epiril368_22C_Rep1_L1.bam
The output from this command gave me unmapped read1 and read2 files which I concatenate as:
cat unmapped.read1.fq unmapped.read2.fq > unmapped.fq

Now when I used epiTeome, it giving me error:

perl epiTEome.pl -gff Analysis/tair10TEs.gff3 -ref Analysis/TAIR10_chr_all.epiTEome.masked.fasta -un trial/Epiril368_4_C_R1_L001_R1_val_1.fq.gz_unmapped_reads_1.fq -t Analysis/teid.lst

Possible precedence issue with control flow operator at epiTEome.pl line 482.
INFO epiTEome.pl Fri Jun 28 09:49:46 2019 Start program!
INFO epiTEome.pl Fri Jun 28 09:49:46 2019 Run Module: readGffFile!

INFO epiTEome.pl Fri Jun 28 09:49:49 2019 STEP 1: read ends mapping.
INFO epiTEome.pl Fri Jun 28 09:49:49 2019 Run Module: splitFastq!

------------- EXCEPTION: Bio::Root::Exception -------------
MSG: Missing sequence and/or quality data; line: 4
STACK: Error::throw
STACK: Bio::Root::Root::throw /usr/local/share/perl/5.26.1/Bio/Root/Root.pm:449
STACK: Bio::SeqIO::fastq::next_dataset /usr/local/share/perl/5.26.1/Bio/SeqIO/fastq.pm:121
STACK: main::splitFastq epiTEome.pl:501
STACK: main::main epiTEome.pl:93
STACK: epiTEome.pl:55

What I understood from the error is I have problem in unmapped.fq file
If you give some suggestions that would be helpful for me.

Thank you!
Regards

CS791