Attempting to Perform MYRNA small example

[cutcopy11]

Hello all,

I am trying to get MyRNA to work on an Ubuntu Linux platform.
Specifically, I could not get the small yeast example to work.

The SRA-tool-kit would not work as was a problem for some other users.

http://seqanswers.com/forums/showthread.php?t=10462

I posted a message on this older thread but did not receive a response. So, I am posting a new thread. I appologize for its length but I want to thoroughly explain my problem, which I try to present in an organized, logical manner.

1)

The test option revealed that I had not installed the sra-tool-kit even though I had.
R was successfully installed and I downloaded / installed the proper packages. See below.

########## Installing R and packages

ckcollings@Anderson-Lab:~$ sudo apt-get update
ckcollings@Anderson-Lab:~$ sudo apt-get install r-base
ckcollings@Anderson-Lab:~$ R
> source("http://bioconductor.org/biocLite.R")
> biocLite()
> biocLite("limma")
> biocLite("IRanges")
> biocLite("biomaRt")
> biocLite("geneplotter")
> install.packages("lmtest", repos="http://cran.r-project.org")
> install.packages("multicore", repos="http://cran.r-project.org")
> q()
Save workspace image? [y/n/c]: n



########## MyRNA test

$ ./myrna_local --test

Summary:
bowtie: INSTALLED at /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie
R: INSTALLED with RHOME at /usr/bin/Rscript
fastq-dump: NOT INSTALLED
PASSED WITH ***WARNING***: SRA toolkit fastq-dump not found; .sra inputs won't work but others will


2)

(I do not show the steps involved in downloading and unzipping the references.)

I
a) downloaded the SRA files in the small manifest files,
b) converted them to fastq,
and c) renamed them.

########## downloading SRA files and converting them to fastq in small yeast example
a)
ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ wget ftp://ftp-trace.ncbi.nih.gov/sra/sra...14335.lite.sra

ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ wget ftp://ftp-trace.ncbi.nih.gov/sra/sra...14339.lite.sra

b)
ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ ../sratoolkit.2.1.6-ubuntu32/fastq-dump ./SRR014335.lite.sra

ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ ../sratoolkit.2.1.6-ubuntu32/fastq-dump ./SRR014339.lite.sra

c)
ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ mv SRR014335.fastq wild-1.fastq

ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ mv SRR014339.fastq rrp6_lsm1_pat1-1.fastq


3)

Now, the tutorial, in my humble, inexperienced opinion, does not really explain how to input actual fastq files.

I tried this but obtained the following error below. Can anyone help me?
Thanks,
Clayton


########## attempting to run MYRNA small yeast example
ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ ./myrna_local --input=wild-1.fastq rrp6_lsm1_pat1-1.fastq --reference=./MYRNA_REFS/yeast_ensembl_61 --output=output_small --cpus=4
(I also tried using the --input option twice, once for each file and got the same result.)

**** I also tried this

ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ ./myrna_local --input=./fastqfiles/ --reference=./MYRNA_REFS/yeast_ensembl_61 --output=output_small --cpus=4
(I put the 2 sra files into a folder named fastqfiles) ... still no luck

Also, the --sra-toolkit option does not work when I try using the small_manifest file with the --preprocess option.

***WARNING***
***WARNING***: SRA toolkit fastq-dump not found; .sra inputs won't work but others will
***WARNING***

Myrna job
------------
Local commands in: /tmp/myrna-ET4Woga0NF/invoke.scripts/myrna.8239.sh
Running...
=== Directory checker ===
Time: 13:07:59 11-Oct-2011
Input: /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq
Output: /home/ckcollings/Desktop/MYRNA_HOME/output_small
Intermediate: /tmp/myrna-ET4Woga0NF/myrna/intermediate/8239
Counters: /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters
Options: [ --force ]
Removing directory /home/ckcollings/Desktop/MYRNA_HOME/output_small due to --force
Removing directory /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters due to --force
==========================
Stage 1 of 6. Align
==========================
Time: 13:07:59 11-Oct-2011
=== Map ===
# parallel mappers: 1
Input: /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq
Output: /tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align
Intermediate: /tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre
Retries / delay: 3 / 5
Options: [ --force ]
Starting 1 mappers with command:
perl /home/ckcollings/Desktop/MYRNA_HOME/Align.pl --bowtie /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie --discard-reads=0 --index-local=/home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61 --partlen=1000000 --qual=phred33 --counters /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt --truncate=0 --globals=/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals --discard-mate=0 --pool-trim-length=0 -- --partition 1000000 --mm -t --hadoopout --startverbose -m 1
Pid 8292 processing input /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq [1 of 1]...
Non-zero return (2304) after executing command 'cat /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq | perl /home/ckcollings/Desktop/MYRNA_HOME/Align.pl --bowtie /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie --discard-reads=0 --index-local=/home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61 --partlen=1000000 --qual=phred33 --counters /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt --truncate=0 --globals=/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals --discard-mate=0 --pool-trim-length=0 -- --partition 1000000 --mm -t --hadoopout --startverbose -m 1 >/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align/map-08292 2>/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre/map.err/map-08292'
Retrying in 5 seconds...
Non-zero return (2304) after executing command 'cat /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq | perl /home/ckcollings/Desktop/MYRNA_HOME/Align.pl --bowtie /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie --discard-reads=0 --index-local=/home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61 --partlen=1000000 --qual=phred33 --counters /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt --truncate=0 --globals=/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals --discard-mate=0 --pool-trim-length=0 -- --partition 1000000 --mm -t --hadoopout --startverbose -m 1 >/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align/map-08292 2>/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre/map.err/map-08292'
Retrying in 5 seconds...
Non-zero return (2304) after executing command 'cat /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq | perl /home/ckcollings/Desktop/MYRNA_HOME/Align.pl --bowtie /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie --discard-reads=0 --index-local=/home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61 --partlen=1000000 --qual=phred33 --counters /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt --truncate=0 --globals=/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals --discard-mate=0 --pool-trim-length=0 -- --partition 1000000 --mm -t --hadoopout --startverbose -m 1 >/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align/map-08292 2>/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre/map.err/map-08292'
Retrying in 5 seconds...
Non-zero return (2304) after executing command 'cat /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq | perl /home/ckcollings/Desktop/MYRNA_HOME/Align.pl --bowtie /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie --discard-reads=0 --index-local=/home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61 --partlen=1000000 --qual=phred33 --counters /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt --truncate=0 --globals=/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals --discard-mate=0 --pool-trim-length=0 -- --partition 1000000 --mm -t --hadoopout --startverbose -m 1 >/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align/map-08292 2>/tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre/map.err/map-08292'
Retrying in 5 seconds...
Fatal error 1.1.2:M130: Out of retries; aborting...

When requesting support, please include the full output printed here.
If a child process was the cause of the error, the output should
include the relevant error message from the child's error log. You may
be asked to provide additional files as well.
******
* Aborting master loop because child 8292 failed
* (other children may also have failed)
* Input file or string was:
* /home/ckcollings/Desktop/MYRNA_HOME/wild-1.fastq:
* Error message is in file: /tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/align.map.pre/map.err/map-08292, also printed below
******
* Align.pl: s3cmd: found: , given:
* Align.pl: jar: found: , given:
* Align.pl: hadoop: found: , given:
* Align.pl: wget: found: /usr/bin/wget, given:
* Align.pl: s3cfg:
* Align.pl: bowtie: found: , given: /home/ckcollings/Desktop/MYRNA_HOME/bin/linux64/bowtie
* Align.pl: partition len: 1000000
* Align.pl: ref:
* Align.pl: quality: phred33
* Align.pl: truncate at: 0
* Align.pl: discard mate: 0
* Align.pl: discard reads < truncate len: 0
* Align.pl: SAM passthrough: 0
* Align.pl: Straight through: 0
* Align.pl: globals directory: /tmp/myrna-ET4Woga0NF/myrna/intermediate/8239/globals
* Align.pl: pool replicates?: 0
* Align.pl: pool trim length: 0
* Align.pl: pool technical replicates?: 0
* Align.pl: local index path: /home/ckcollings/Desktop/MYRNA_HOME/MYRNA_REFS/yeast_ensembl_61/index/yeast_ensembl_61
* Align.pl: counters: /home/ckcollings/Desktop/MYRNA_HOME/output_small_counters/counters.txt
* Align.pl: dest dir:
* Align.pl: bowtie args: --partition 1000000 --mm -t --hadoopout --startverbose -m 1
* Align.pl: ls -al
* Align.pl: total 8
* drwxr-xr-x 2 ckcollings ckcollings 4096 2011-10-11 13:07 .
* drwxr-xr-x 3 ckcollings ckcollings 4096 2011-10-11 13:07 ..
* Align.pl: Retrived 0 counters from previous stages
* Align.pl: Read first line of stdin:
* @SRR014335.1 GA-EAS46_1_209DH:5:1:889:471 length=36
* Bad number of read tokens ; expected 3 or 5:
* @SRR014335.1 GA-EAS46_1_209DH:5:1:889:471 length=36
******
Fatal error 1.1.2:M140: Aborting because child with PID 8292 exited abnormally

When requesting support, please include the full output printed here.
If a child process was the cause of the error, the output should
include the relevant error message from the child's error log. You may
be asked to provide additional files as well.
Non-zero exitlevel from Align stage

[cutcopy11]

I forgot to add --quality solexa64 but I still could not get it to work

ckcollings@Anderson-Lab:~/Desktop/MYRNA_HOME$ ./myrna_local --input=./fastqfiles --reference=./MYRNA_REFS/yeast_ensembl_61 --quality solexa64 --output=output_small --cpus=4 

[axiom7]

Hi, Did you ever figure this out. I am having the exact same problem.
Thanks.
Susan

[narain]

I am getting the same problem

Bad number of read tokens ; expected 3 or 5

[9000revs]

Once you installed SRA toolkit, did you set the env variable called MYRNA_SRATOOLKIT_HOME?