error blast

[frankbioinfo]

I am using the example file from previous issue that you had uploaded (exampleVar.txt) and config file as described using hg19 fasta i get this error

2017-05-16 17:18:11.326957 Primers to test: 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.332841 potUnique 0.01 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.338627 potUnique 0.1 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.344542 potUnique 1 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.350145 potUnique 10 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.355408 potUnique 100 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.361081 potUnique 10 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-16 17:18:11.366995 potUnique 100 0
unique primers: 0
non-unique primers: 0
forbidden primers: 0
2017-05-16 17:18:11.372512

Not sure what it means

[zichner]

Can you please upload the config file you used and post the command you use to run the pipeline.
This might help to identify the problem. Thanks!

[frankbioinfo]

PrimerDesign 2.cfg.zip
exampleVar (1).txt

command:
python primerDesign.py PrimerDesign.2.cfg exampleVar.txt results.txt

[frankbioinfo]

does it help?

[zichner]

Thanks for the files. I am currently looking into the issue.
Can you please also post the full program output (including the part before the error). Thanks.

[zichner]

The fact that your output says Primers to test: 0 indicates that there might have been a problem with the primer3 step and therefore, no primers were predicted. Was there any error message related to primer3?

[frankbioinfo]

No error related to primer3. but here is weird thing I get an output file but still see these error (attached result out file and the full error file. (can i trust the output)
primerdesign_full report.txt
)

Number of variants left: 1
2017-05-17 11:26:09.789408 Primers to test: 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.795314 potUnique 0.01 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.801093 potUnique 0.1 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.807209 potUnique 1 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.813552 potUnique 10 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.819844 potUnique 100 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.825203 potUnique 10 0
[blast] ERROR: Length of a discontiguous word template (contiguous word if 0) [ask] is bad or out of range [? to ?]
2017-05-17 11:26:09.830575 potUnique 100 0
unique primers: 176
non-unique primers: 0
forbidden primers: 0
results_out.txt

Thank you for looking in to this, appreciate it.

[zichner]

The full output shows that the BLAST step (checking whether primers are unique) is not working.
As a consequence, the results you get are not correct (the primers are valid but they are not necessarily specific).

In order to make the usage of primerDesign much easier, I now build a Docker container that contains all necessary software tools as well as a BLAST database for human. This should solve the problems. It would be great if you could try it. You can find all details in the README.md file.
Please note: The BLAST DB used in the Docker container is based on the human reference genome version hs37d5 (provided by the 1000 genomes project). As a consequence, chromosome names must not contain chr (see the exampleVariants.txt file).

[frankbioinfo]

Ah ha!, the docker to rescue again! thank you docker should solve most of the issues. will keep you posted.

[frankbioinfo]

@zichner is this correct? using stringent25bp and example file as is please see below log and error
docker pull zichner/primer-design
Using default tag: latest

latest: Pulling from zichner/primer-design
b6f892c0043b: Pull complete
55010f332b04: Pull complete
2955fb827c94: Pull complete
3deef3fcbd30: Pull complete
cf9722e506aa: Pull complete
5a082b0edddc: Pull complete
ad8eeb4b89c8: Pull complete
f7f62b705823: Pull complete
93b32fb35380: Pull complete
270f1435739b: Pull complete
3596da27f287: Pull complete
Digest: sha256:20e5448e1ab5643907cea7e2d3c7a07fce7bde820d2d75029d415fdc0247fd6d
Status: Downloaded newer image for zichner/primer-design:latest

#created a folder primer_design within documents.

docker run -v /Users/s/Documents/primer_design/ zichner/primer-design /Users/s/Documents/primer_design/primer_design_config_docker.cfg /Users/s/Documents/primer_design/examplevariants_docker.txt /Users/s/Documents/primer_design/results.txt
2017-05-31 15:24:15.968219
Traceback (most recent call last):
File "/opt/primerDesign/primerDesign.py", line 471, in
main()
File "/opt/primerDesign/primerDesign.py", line 24, in main
maxSeqDist = config.getint("PrimerDesign", "max_dist_for_sequencing")
File "/usr/lib/python2.7/ConfigParser.py", line 359, in getint
return self._get(section, int, option)
File "/usr/lib/python2.7/ConfigParser.py", line 356, in _get
return conv(self.get(section, option))
File "/usr/lib/python2.7/ConfigParser.py", line 607, in get
raise NoSectionError(section)
ConfigParser.NoSectionError: No section: 'PrimerDesign'

[frankbioinfo]

Docker run with multiple options
#1
docker run zichner/primer-design python primerDesign.py
2017-05-31 16:16:44.930912
Traceback (most recent call last):
File "/opt/primerDesign/primerDesign.py", line 471, in
main()
File "/opt/primerDesign/primerDesign.py", line 24, in main
maxSeqDist = config.getint("PrimerDesign", "max_dist_for_sequencing")
File "/usr/lib/python2.7/ConfigParser.py", line 359, in getint
return self._get(section, int, option)
File "/usr/lib/python2.7/ConfigParser.py", line 356, in _get
return conv(self.get(section, option))
File "/usr/lib/python2.7/ConfigParser.py", line 607, in get
raise NoSectionError(section)

#2
docker run zichner/primer-design python /opt/primerDesign/primerDesign.py
2017-05-31 16:18:11.307375
Traceback (most recent call last):
File "/opt/primerDesign/primerDesign.py", line 471, in
main()
File "/opt/primerDesign/primerDesign.py", line 24, in main
maxSeqDist = config.getint("PrimerDesign", "max_dist_for_sequencing")
File "/usr/lib/python2.7/ConfigParser.py", line 359, in getint
return self._get(section, int, option)
File "/usr/lib/python2.7/ConfigParser.py", line 356, in _get
return conv(self.get(section, option))
File "/usr/lib/python2.7/ConfigParser.py", line 607, in get
raise NoSectionError(section)
ConfigParser.NoSectionError: No section: 'PrimerDesign'

#3
docker run zichner/primer-design
2017-05-31 16:18:53.189541
Traceback (most recent call last):
File "/opt/primerDesign/primerDesign.py", line 471, in
main()
File "/opt/primerDesign/primerDesign.py", line 23, in main
config.read(sys.argv[1])
IndexError: list index out of range

[zichner]

When you use a docker container and want to access files from your normal file system, you have to map a directory of your file system to a directory inside the docker container. Afterwards you can access the files as if they were inside the container (ie., you have to use the directory structure as it is in the container).

So, in your case the command should be:

docker run -v /Users/s/Documents/primer_design:/data/ zichner/primer-design /data/primer_design_config_docker.cfg /data/examplevariants_docker.txt /data/results.txt

However, /data/ is specified as the working directory in the container, so you can omit it in the file paths and just do:

docker run -v /Users/s/Documents/primer_design:/data/ zichner/primer-design primer_design_config_docker.cfg examplevariants_docker.txt results.txt

Both commands assume that you have the files primer_design_config_docker.cfg and examplevariants_docker.txt in the directory /Users/s/Documents/primer_design.

I hope this helps.

[frankbioinfo]

results.txt
Option1 worked! Thanks attached result and log, any red flags?
primerdesign.log.txt

[frankbioinfo]

Thank you for your help, I have one last question what was your approach to build the example variant file do you have a snippet of the input and output for that file if you do not mind educating me. Appreciate your help.