Differential expression error

[jfad1286]

Hi. I was wondering if you could help me. I have some RNA Seq data that I wanted to analyse and so far I've managed to follow the guide all the way to the differential expression analysis. I get an error and I'm not sure how to solve it. I attach the log from the analysis error.

I have data from 6 animals, 3 wild type (WT) and 3 knock-outs (KO). I created the groups for both with three animals in each. Then I created another group with both WT and KO groups inside, this allowed me to select the "sample group" (their genotype in this case) as the contrast variables. I left all other options in their default settings, although because of the error I also tried changing different options, but I haven't been able to make it work.

Could the way I created the analysis groups be the issue, how can I solve it if it is?

Thanks for your help!

Juan

Error.txt

[alaimos]

Dear Juan,

Thank you for the report. I need to run some tests to determine the exact source of your problem.
I will let you know if I need more details about the analysis you are trying to run in order to determine the issue.

[alaimos]

I traced the problem to a corrupted R package. As a result, I've updated and rebuilt the RNAdetector docker container.
Next time, when you start the app with an internet connection, you should be notified of a new update (the download is automatic).

Please, let me know if this solves your issue.

[jfad1286]

Dear Salvatore, Thanks for your help! I started the application and it automatically downloaded the update, but I still have an error. I attach the log. All the best, Juan

On 24 Aug 2021, 18:46 +0100, Salvatore Alaimo ***@***.***>, wrote: I traced the problem to a corrupted R package. As a result, I've updated and rebuilt the RNAdetector docker container. Next time, when you start the app with an internet connection, you should be notified of a new update (the download is automatic). Please, let me know if this solves your issue. — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub, or unsubscribe. Triage notifications on the go with GitHub Mobile for iOS or Android.

Logs of DEG WT-KO Starting diffential expression analysis. Loading required package: VennDiagram Loading required package: grid Attaching package: ÔgridÕ The following object is masked from Ôpackage:BiostringsÕ: pattern Loading required package: futile.logger 2021-08-26 16:39:53: Data processing started... Read counts file: imported custom data frame Conditions: KO, WT Samples to include: D1, C21, F12, F11 Samples to exclude: none Requested contrasts: WT_vs_KO Annotation: embedded Organism: custom Reference source: ensembl Count type: gene Transcriptional level: gene Exon filters: min.active.exons min.active.exons: exons.per.gene: 5 min.exons: 2 frac: 0.2 Gene filters: length, avg.reads, expression length: length: 500 avg.reads: average.per.bp: 100 quantile: 0.75 expression: median: 1 mean: 0 custom: NA Filter application: prenorm Normalization algorithm: edger Normalization arguments: method: TMM logratioTrim: 0.3 sumTrim: 0.05 doWeighting: TRUE Acutoff: -1e+10 p: 0.75 Statistical algorithm: limma, deseq Statistical arguments: limma: none deseq: parametric Meta-analysis method: simes Multiple testing correction: qvalue p-value threshold: 0.05 Logarithmic transformation offset: 1 Quality control plots: mds, readnoise, correl, pairwise, boxplot, deheatmap, volcano, venn Figure format: png, pdf Output directory: /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko Output data: annotation, p.value, adj.p.value, meta.p.value, adj.meta.p.value, fold.change, stats Output scale(s): natural, log2 Output values: raw, normalized Output statistics: mean, median, sd Saving gene model to /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko/data/gene_model.RData Removing genes with zero counts in all samples... Prefiltering normalization with: edger Applying gene filter length... Threshold below which ignored: 500 Applying gene filter avg.reads...Error in quantile.default(newX[, i], ...) : missing values and NaN's not allowed if 'na.rm' is FALSE Execution halted Error: Symfony\Component\Process\Exception\ProcessFailedException: The command "'Rscript' '/rnadetector/scripts/de_analysis.R' '-c' '/rnadetector/ws/storage/app/public/jobs/9/deg_config_deg-wt-ko.json'" failed. Exit Code: 1(General error) Working directory: /rnadetector/ws/storage/app/public/jobs/9 Output: ================ Error Output: ================ Loading required package: VennDiagram Loading required package: grid Attaching package: ÔgridÕ The following object is masked from Ôpackage:BiostringsÕ: pattern Loading required package: futile.logger 2021-08-26 16:39:53: Data processing started... Read counts file: imported custom data frame Conditions: KO, WT Samples to include: D1, C21, F12, F11 Samples to exclude: none Requested contrasts: WT_vs_KO Annotation: embedded Organism: custom Reference source: ensembl Count type: gene Transcriptional level: gene Exon filters: min.active.exons min.active.exons: exons.per.gene: 5 min.exons: 2 frac: 0.2 Gene filters: length, avg.reads, expression length: length: 500 avg.reads: average.per.bp: 100 quantile: 0.75 expression: median: 1 mean: 0 custom: NA Filter application: prenorm Normalization algorithm: edger Normalization arguments: method: TMM logratioTrim: 0.3 sumTrim: 0.05 doWeighting: TRUE Acutoff: -1e+10 p: 0.75 Statistical algorithm: limma, deseq Statistical arguments: limma: none deseq: parametric Meta-analysis method: simes Multiple testing correction: qvalue p-value threshold: 0.05 Logarithmic transformation offset: 1 Quality control plots: mds, readnoise, correl, pairwise, boxplot, deheatmap, volcano, venn Figure format: png, pdf Output directory: /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko Output data: annotation, p.value, adj.p.value, meta.p.value, adj.meta.p.value, fold.change, stats Output scale(s): natural, log2 Output values: raw, normalized Output statistics: mean, median, sd Saving gene model to /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko/data/gene_model.RData Removing genes with zero counts in all samples... Prefiltering normalization with: edger Applying gene filter length... Threshold below which ignored: 500 Applying gene filter avg.reads...Error in quantile.default(newX[, i], ...) : missing values and NaN's not allowed if 'na.rm' is FALSE Execution halted in /rnadetector/ws/app/Utils.php:51 Stack trace: #0 /rnadetector/ws/app/Jobs/Types/AbstractJob.php(443): App\Utils::runCommand() #1 /rnadetector/ws/app/Jobs/Types/DiffExprAnalysisJobType.php(564): App\Jobs\Types\AbstractJob::runCommand() #2 /rnadetector/ws/app/Jobs/Request.php(72): App\Jobs\Types\DiffExprAnalysisJobType->handle() #3 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(36): App\Jobs\Request->handle() #4 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Util.php(37): Illuminate\Container\BoundMethod::Illuminate\Container\{closure}() #5 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(93): Illuminate\Container\Util::unwrapIfClosure() #6 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(37): Illuminate\Container\BoundMethod::callBoundMethod() #7 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Container.php(590): Illuminate\Container\BoundMethod::call() #8 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Bus/Dispatcher.php(94): Illuminate\Container\Container->call() #9 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(130): Illuminate\Bus\Dispatcher->Illuminate\Bus\{closure}() #10 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(105): Illuminate\Pipeline\Pipeline->Illuminate\Pipeline\{closure}() #11 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Bus/Dispatcher.php(98): Illuminate\Pipeline\Pipeline->then() #12 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(83): Illuminate\Bus\Dispatcher->dispatchNow() #13 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(130): Illuminate\Queue\CallQueuedHandler->Illuminate\Queue\{closure}() #14 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(105): Illuminate\Pipeline\Pipeline->Illuminate\Pipeline\{closure}() #15 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(85): Illuminate\Pipeline\Pipeline->then() #16 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(59): Illuminate\Queue\CallQueuedHandler->dispatchThroughMiddleware() #17 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Jobs/Job.php(88): Illuminate\Queue\CallQueuedHandler->call() #18 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(368): Illuminate\Queue\Jobs\Job->fire() #19 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(314): Illuminate\Queue\Worker->process() #20 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(134): Illuminate\Queue\Worker->runJob() #21 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Console/WorkCommand.php(112): Illuminate\Queue\Worker->daemon() #22 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Console/WorkCommand.php(96): Illuminate\Queue\Console\WorkCommand->runWorker() #23 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(36): Illuminate\Queue\Console\WorkCommand->handle() #24 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Util.php(37): Illuminate\Container\BoundMethod::Illuminate\Container\{closure}() #25 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(93): Illuminate\Container\Util::unwrapIfClosure() #26 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(37): Illuminate\Container\BoundMethod::callBoundMethod() #27 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Container.php(590): Illuminate\Container\BoundMethod::call() #28 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Command.php(134): Illuminate\Container\Container->call() #29 /rnadetector/ws/vendor/symfony/console/Command/Command.php(255): Illuminate\Console\Command->execute() #30 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Command.php(121): Symfony\Component\Console\Command\Command->run() #31 /rnadetector/ws/vendor/symfony/console/Application.php(1009): Illuminate\Console\Command->run() #32 /rnadetector/ws/vendor/symfony/console/Application.php(273): Symfony\Component\Console\Application->doRunCommand() #33 /rnadetector/ws/vendor/symfony/console/Application.php(149): Symfony\Component\Console\Application->doRun() #34 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Application.php(93): Symfony\Component\Console\Application->run() #35 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Foundation/Console/Kernel.php(131): Illuminate\Console\Application->run() #36 /rnadetector/ws/artisan(37): Illuminate\Foundation\Console\Kernel->handle() #37 {main} Next App\Exceptions\ProcessingJobException: The command "'Rscript' '/rnadetector/scripts/de_analysis.R' '-c' '/rnadetector/ws/storage/app/public/jobs/9/deg_config_deg-wt-ko.json'" failed. Exit Code: 1(General error) Working directory: /rnadetector/ws/storage/app/public/jobs/9 Output: ================ Error Output: ================ Loading required package: VennDiagram Loading required package: grid Attaching package: ÔgridÕ The following object is masked from Ôpackage:BiostringsÕ: pattern Loading required package: futile.logger 2021-08-26 16:39:53: Data processing started... Read counts file: imported custom data frame Conditions: KO, WT Samples to include: D1, C21, F12, F11 Samples to exclude: none Requested contrasts: WT_vs_KO Annotation: embedded Organism: custom Reference source: ensembl Count type: gene Transcriptional level: gene Exon filters: min.active.exons min.active.exons: exons.per.gene: 5 min.exons: 2 frac: 0.2 Gene filters: length, avg.reads, expression length: length: 500 avg.reads: average.per.bp: 100 quantile: 0.75 expression: median: 1 mean: 0 custom: NA Filter application: prenorm Normalization algorithm: edger Normalization arguments: method: TMM logratioTrim: 0.3 sumTrim: 0.05 doWeighting: TRUE Acutoff: -1e+10 p: 0.75 Statistical algorithm: limma, deseq Statistical arguments: limma: none deseq: parametric Meta-analysis method: simes Multiple testing correction: qvalue p-value threshold: 0.05 Logarithmic transformation offset: 1 Quality control plots: mds, readnoise, correl, pairwise, boxplot, deheatmap, volcano, venn Figure format: png, pdf Output directory: /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko Output data: annotation, p.value, adj.p.value, meta.p.value, adj.meta.p.value, fold.change, stats Output scale(s): natural, log2 Output values: raw, normalized Output statistics: mean, median, sd Saving gene model to /rnadetector/ws/storage/app/public/jobs/9/deg_report_deg-wt-ko/data/gene_model.RData Removing genes with zero counts in all samples... Prefiltering normalization with: edger Applying gene filter length... Threshold below which ignored: 500 Applying gene filter avg.reads...Error in quantile.default(newX[, i], ...) : missing values and NaN's not allowed if 'na.rm' is FALSE Execution halted in /rnadetector/ws/app/Utils.php:78 Stack trace: #0 /rnadetector/ws/app/Jobs/Types/AbstractJob.php(445): App\Utils::mapCommandException() #1 /rnadetector/ws/app/Jobs/Types/DiffExprAnalysisJobType.php(564): App\Jobs\Types\AbstractJob::runCommand() #2 /rnadetector/ws/app/Jobs/Request.php(72): App\Jobs\Types\DiffExprAnalysisJobType->handle() #3 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(36): App\Jobs\Request->handle() #4 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Util.php(37): Illuminate\Container\BoundMethod::Illuminate\Container\{closure}() #5 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(93): Illuminate\Container\Util::unwrapIfClosure() #6 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(37): Illuminate\Container\BoundMethod::callBoundMethod() #7 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Container.php(590): Illuminate\Container\BoundMethod::call() #8 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Bus/Dispatcher.php(94): Illuminate\Container\Container->call() #9 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(130): Illuminate\Bus\Dispatcher->Illuminate\Bus\{closure}() #10 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(105): Illuminate\Pipeline\Pipeline->Illuminate\Pipeline\{closure}() #11 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Bus/Dispatcher.php(98): Illuminate\Pipeline\Pipeline->then() #12 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(83): Illuminate\Bus\Dispatcher->dispatchNow() #13 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(130): Illuminate\Queue\CallQueuedHandler->Illuminate\Queue\{closure}() #14 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Pipeline/Pipeline.php(105): Illuminate\Pipeline\Pipeline->Illuminate\Pipeline\{closure}() #15 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(85): Illuminate\Pipeline\Pipeline->then() #16 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/CallQueuedHandler.php(59): Illuminate\Queue\CallQueuedHandler->dispatchThroughMiddleware() #17 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Jobs/Job.php(88): Illuminate\Queue\CallQueuedHandler->call() #18 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(368): Illuminate\Queue\Jobs\Job->fire() #19 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(314): Illuminate\Queue\Worker->process() #20 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Worker.php(134): Illuminate\Queue\Worker->runJob() #21 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Console/WorkCommand.php(112): Illuminate\Queue\Worker->daemon() #22 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Queue/Console/WorkCommand.php(96): Illuminate\Queue\Console\WorkCommand->runWorker() #23 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(36): Illuminate\Queue\Console\WorkCommand->handle() #24 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Util.php(37): Illuminate\Container\BoundMethod::Illuminate\Container\{closure}() #25 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(93): Illuminate\Container\Util::unwrapIfClosure() #26 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/BoundMethod.php(37): Illuminate\Container\BoundMethod::callBoundMethod() #27 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Container/Container.php(590): Illuminate\Container\BoundMethod::call() #28 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Command.php(134): Illuminate\Container\Container->call() #29 /rnadetector/ws/vendor/symfony/console/Command/Command.php(255): Illuminate\Console\Command->execute() #30 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Command.php(121): Symfony\Component\Console\Command\Command->run() #31 /rnadetector/ws/vendor/symfony/console/Application.php(1009): Illuminate\Console\Command->run() #32 /rnadetector/ws/vendor/symfony/console/Application.php(273): Symfony\Component\Console\Application->doRunCommand() #33 /rnadetector/ws/vendor/symfony/console/Application.php(149): Symfony\Component\Console\Application->doRun() #34 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Console/Application.php(93): Symfony\Component\Console\Application->run() #35 /rnadetector/ws/vendor/laravel/framework/src/Illuminate/Foundation/Console/Kernel.php(131): Illuminate\Console\Application->run() #36 /rnadetector/ws/artisan(37): Illuminate\Foundation\Console\Kernel->handle() #37 {main}

[YK791]

Dear Juan,
I wonder whether you have solved the problem?
I experience exactly the same.
Thanks,
Yulia