Issue with fusion detection

[chetanya]

Hi,

I would like to use TRUP to detect chimeric fusions in my RNA-Seq datasets. Unfortunately, I keep running into the following issue:

RTrace.pl --runlevel 4 --sampleName sample --seqType p --readpool /sc/orga/scratch/pandyc01/TRUP/Data --root /sc/orga/scratch/pandyc01/TRUP --threads 4 --anno /sc/orga/projects/PBG/REFERENCES/hg19/TRUP/hg19

[2015/6/26 11:1:13] ####### lane name is set to sample #######

lanefile: /sc/orga/scratch/pandyc01/TRUP/Data/sample_R1.fq.gz
lanefile: /sc/orga/scratch/pandyc01/TRUP/Data/sample_R2.fq.gz

[2015/6/26 11:1:13] ####### preparing directories #######

read length and trimed length are not set, will take the original read length (100 bp) for both (no trimming).

[2015/6/26 11:1:13] ####### insert mean and sd calculation #######

qual_min: 2; qual_max: 41; qual_zero: 35; qual_shift: -2.
Error in setwd(path) : cannot change working directory
Execution halted
Use of uninitialized value $number in int at /hpc/packages/minerva-common/trup/0.1/TRUP/bin/RTrace.pl line 2064.
Use of uninitialized value $number in numeric ge (>=) at /hpc/packages/minerva-common/trup/0.1/TRUP/bin/RTrace.pl line 2065.
Use of uninitialized value $insert_sd in substitution (s///) at /hpc/packages/minerva-common/trup/0.1/TRUP/bin/RTrace.pl line 525.
Use of uninitialized value $number in int at /hpc/packages/minerva-common/trup/0.1/TRUP/bin/RTrace.pl line 2064.
Use of uninitialized value $number in numeric ge (>=) at /hpc/packages/minerva-common/trup/0.1/TRUP/bin/RTrace.pl line 2065.
insert mean: -200 insert_sd: 0
two mates is overlapping too much, please trim more.

[chetanya]

My current environment:

1. zlib/1.2.8 5) gmp/5.1.3 9) star/2.4.1c 13) gsl/1.16 17) torque/4.1.5-test
2. samtools/1.1 6) mpfr/3.1.2 10) velvet/1.1.06 14) geos/3.4.2 18) openmpi/1.6.5
3. bowtie2/2.1.0 7) mpc/1.0.2 11) oases/0.2.09 15) netcdf/4.3.3.1 19) R/3.0.3(default)
4. blat/3.0.5 8) gcc/4.8.2 12) intel/parallel_studio_xe_2015 16) lsf/9.1 20) trup/0.1

[chetanya]

If I understand the code correctly, looks like the "fragment_length.R" script is having issues with the input files. Do you mind looking into this as soon as possible?

[ruping]

Hi, would you mind sending me a small part of your sequencing file
(fastq) so I can test it? I have run the pipeline thousands of times
without error like that.

simply do "head -400000 fastq_1.fq >sendme_1.fq" and "head -400000
fastq_2.fq >sendme_2.fq"

gzip sendme_1.fq and sendme_2.fq

then send me.

Thanks.

Ruping,

On 7/31/2015 10:41 AM, Chetanya Pandya wrote:

If I understand the code correctly, looks like the "fragment_length.R"
script is having issues with the input files. Do you mind looking into
this as soon as possible?

—
Reply to this email directly or view it on GitHub
#3 (comment).

^ Sun,Ruping ####################^

Curtis Lab,

Stanford Cancer Institute,

Stanford University,

780 Welch Rd, office-CJ220C01

Palo Alto, CA 94304

Email: ruping@stanford.edu

@################################@

[chetanya]

Hi Ruping,

You can access the files here:
https://drive.google.com/file/d/0B8cD1iIefQ6Bc2V1SVJkbjhrOTA/view?usp=sharing
https://drive.google.com/file/d/0B8cD1iIefQ6BenE0Y084VVBXRVU/view?usp=sharing

The commands I'm running:
cd /sc/orga/scratch/pandyc01/TRUP

RTrace.pl --runlevel 1 --sampleName sample --seqType p --readpool /sc/orga/scratch/pandyc01/TRUP/Data --root /sc/orga/scratch/pandyc01/TRUP --threads 4 --anno /sc/orga/projects/PBG/REFERENCES/hg19/TRUP/hg19 --QC

RTrace.pl --runlevel 4 --sampleName sample --seqType p --readpool /sc/orga/scratch/pandyc01/TRUP/Data --root /sc/orga/scratch/pandyc01/TRUP --threads 4 --anno /sc/orga/projects/PBG/REFERENCES/hg19/TRUP/hg19 --RA 1

Look forward to hearing from you.

Best,
Chetanya

[zhaoming159753]

Hi Ruping,
i am using TRUP for RNA-seq analysis and i got the same error with Chetanya.

/lustre/home/medzm/software/TRUP-master/bin/RTrace.pl --runlevel 1 --sampleName 229a4961-f7eb-44ba-8f63-0021495585e8 --seqType p --readpool /lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/BLCA/229a4961-f7eb-44ba-8f63-0021495585e8 --root /lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/TRUP_Pepline --threads 8 --anno /lustre/home/medzm/annotation/TRUP_ann

[2015/8/27 17:37:10] ####### lane name is set to 229a4961-f7eb-44ba-8f63-0021495585e8 #######

lanefile: /lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/BLCA/229a4961-f7eb-44ba-8f63-0021495585e8/229a4961-f7eb-44ba-8f63-0021495585e8_1.fq.gz
lanefile: /lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/BLCA/229a4961-f7eb-44ba-8f63-0021495585e8/229a4961-f7eb-44ba-8f63-0021495585e8_2.fq.gz

[2015/8/27 17:37:10] ####### preparing directories #######

read length and trimed length are not set, will take the original read length (48 bp) for both (no trimming).

[2015/8/27 17:37:10] ####### runlevel 1 now #######

total number of read pairs: 5329758

[2015/8/27 17:37:15] perl /lustre/home/medzm/software/TRUP-master/bin//spike_in.pl /lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/TRUP_Pepline/229a4961-f7eb-44ba-8f63-0021495585e8/00_TEST/229a4961-f7eb-44ba-8f63-0021495585e8.spikedin.hits >/lustre/home/medzm/data/TCGA_WGS/TCGA_WGS_RNA-seq/TRUP_Pepline/229a4961-f7eb-44ba-8f63-0021495585e8/00_TEST/229a4961-f7eb-44ba-8f63-0021495585e8.spikedin.fragmentlength

[2015/8/27 17:37:16] ####### runlevel 1 done #######

[2015/8/27 17:37:16] ####### insert mean and sd calculation #######

qual_min: 2; qual_max: 41; qual_zero: 35; qual_shift: -2.
Error in read.table(file = file, header = header, sep = sep, quote = quote, :
no lines available in input
Calls: read.delim -> read.table
Execution halted
Use of uninitialized value $number in int at /lustre/home/medzm/software/TRUP-master/bin/RTrace.pl line 2064.
Use of uninitialized value $number in numeric ge (>=) at /lustre/home/medzm/software/TRUP-master/bin/RTrace.pl line 2065.
Use of uninitialized value $insert_sd in substitution (s///) at /lustre/home/medzm/software/TRUP-master/bin/RTrace.pl line 525.
Use of uninitialized value $number in int at /lustre/home/medzm/software/TRUP-master/bin/RTrace.pl line 2064.
Use of uninitialized value $number in numeric ge (>=) at /lustre/home/medzm/software/TRUP-master/bin/RTrace.pl line 2065.
insert mean: -96 insert_sd: 0
two mates is overlapping too much, please trim more.

what's my problem?

Look forward to hearing from you.

Best Regards;

             Ming