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RNASeq-NF is an NGS analysis pipeline for RNA expression quantification.

License: MIT License

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toolsvanbox ffmmulder tilschaef jvandinter ywilke ssyamoako xclaws emmadebayos

rnaseq-nf's Issues

Warnings and errors

Hi Tilman

I tried to run the pipeline with this script:

#!/bin/bash

module load Java/1.8.0_60
/hpc/pmc_vanboxtel/tools/nextflow/nextflow run /hpc/pmc_vanboxtel/tools/RNASeq-NF/main.nf \
-c run.config \
--fastq_path /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/fastq/SRR1028343/ \
--out_dir ./ \
 -profile slurm

But it gives me some warnings and errors (see below). Can you help me?

Thanks
Mark

Mar-31 11:42:57.644 [main] DEBUG nextflow.cli.Launcher - $> nextflow run /hpc/pmc_vanboxtel/tools/RNASeq-NF/main.nf -c run.config --fastq_path /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/fastq/SRR1028343/ --out_dir ./ -profile slurm
Mar-31 11:43:01.446 [main] INFO  nextflow.cli.CmdRun - N E X T F L O W  ~  version 20.03.0-edge
Mar-31 11:43:02.142 [main] INFO  nextflow.cli.CmdRun - Launching `/hpc/pmc_vanboxtel/tools/RNASeq-NF/main.nf` [loving_feynman] - revision: 1a9a31d90a
Mar-31 11:43:02.939 [main] DEBUG nextflow.config.ConfigBuilder - Found config base: /hpc/pmc_vanboxtel/tools/RNASeq-NF/nextflow.config
Mar-31 11:43:03.040 [main] DEBUG nextflow.config.ConfigBuilder - User config file: /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/RNASeq-NF/run.config
Mar-31 11:43:03.040 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /hpc/pmc_vanboxtel/tools/RNASeq-NF/nextflow.config
Mar-31 11:43:03.041 [main] DEBUG nextflow.config.ConfigBuilder - Parsing config file: /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/RNASeq-NF/run.config
Mar-31 11:43:04.441 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `slurm`
Mar-31 11:43:17.350 [main] DEBUG nextflow.config.ConfigBuilder - Applying config profile: `slurm`
Mar-31 11:43:34.541 [main] DEBUG nextflow.config.ConfigBuilder - Available config profiles: [sge, slurm]
Mar-31 11:43:35.850 [main] DEBUG nextflow.Session - Session uuid: 87fa9927-fbab-4b17-baee-530136b2bb8e
Mar-31 11:43:35.851 [main] DEBUG nextflow.Session - Run name: loving_feynman
Mar-31 11:43:35.941 [main] DEBUG nextflow.Session - Executor pool size: 32
Mar-31 11:43:36.440 [main] DEBUG nextflow.cli.CmdRun - 
  Version: 20.03.0-edge build 5288
  Created: 28-03-2020 11:10 UTC (12:10 CEST)
  System: Linux 3.10.0-1062.4.3.el7.x86_64
  Runtime: Groovy 2.5.8 on Java HotSpot(TM) 64-Bit Server VM 1.8.0_60-b27
  Encoding: UTF-8 (UTF-8)
  Process: [email protected] [143.121.243.143]
  CPUs: 32 - Mem: 125.7 GB (4.2 GB) - Swap: 8 GB (4.8 GB)
Mar-31 11:43:37.444 [main] DEBUG nextflow.Session - Work-dir: /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/RNASeq-NF/work [nfs]
Mar-31 11:43:38.840 [main] DEBUG nextflow.Session - Observer factory: TowerFactory
Mar-31 11:43:39.144 [main] DEBUG nextflow.Session - Observer factory: DefaultObserverFactory
Mar-31 11:43:48.049 [main] DEBUG nextflow.Session - Session start invoked
Mar-31 11:43:48.246 [main] DEBUG nextflow.trace.TraceFileObserver - Flow starting -- trace file: /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/RNASeq-NF/log/nextflow_trace.txt
Mar-31 11:44:03.341 [main] WARN  nextflow.NextflowMeta$Preview - DSL 2 IS AN EXPERIMENTAL FEATURE UNDER DEVELOPMENT -- SYNTAX MAY CHANGE IN FUTURE RELEASE
Mar-31 11:44:03.544 [main] DEBUG nextflow.script.ScriptRunner - > Launching execution
Mar-31 11:44:04.244 [main] WARN  nextflow.script.IncludeDef - Anonymous module inclusion is deprecated -- Replace `include './NextflowModules/Utils/fastq.nf'` with `include { MODULE_NAME } from './NextflowModules/Utils/fastq.nf'`
Mar-31 11:44:20.441 [main] WARN  n.script.WorkflowParamsResolver - Workflow `get` is deprecated -- Use `take` instead
Mar-31 11:44:52.840 [main] WARN  nextflow.script.ScriptBinding - Access to undefined parameter `hts_count_type` -- Initialise it to a default value eg. `params.hts_count_type = some_value`
Mar-31 11:45:15.840 [main] DEBUG nextflow.Session - Workflow process names [dsl2]: BaseRecalibration, SplitIntervals, MultiQC, GatherBaseRecalibrationTables, Lc_extrap, Index, mergeHtseqCounts, Count, MarkDup, mergeFastqLanes, Quant, AlignReads, rpkm, featureCounts, MergeBams, HaplotypeCaller, RSeQC, Fastp, BaseRecalibrationTable, MergeVCFs, SplitNCigarReads, VariantFiltration
Mar-31 11:45:17.645 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:Fastp_0_14_1` matches label `Fastp_0_14_1` for process with name Fastp
Mar-31 11:45:17.655 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: slurm
Mar-31 11:45:17.655 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'slurm'
Mar-31 11:45:17.746 [main] DEBUG nextflow.executor.Executor - [warm up] executor > slurm
Mar-31 11:45:17.847 [main] DEBUG n.processor.TaskPollingMonitor - Creating task monitor for executor 'slurm' > capacity: 1000; pollInterval: 30s; dumpInterval: 5m 
Mar-31 11:45:17.943 [main] DEBUG n.executor.AbstractGridExecutor - Creating executor 'slurm' > queue-stat-interval: 5m
Mar-31 11:45:19.741 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:STAR_2_6_0c_AlignReads` matches label `STAR_2_6_0c_AlignReads` for process with name AlignReads
Mar-31 11:45:19.745 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: slurm
Mar-31 11:45:19.745 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'slurm'
Mar-31 11:45:20.140 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:SAMBAMBA_0_6_8_Index` matches label `SAMBAMBA_0_6_8_Index` for process with name Index
Mar-31 11:45:20.240 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: slurm
Mar-31 11:45:20.240 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'slurm'
Mar-31 11:45:20.642 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:RSeQC_3_0_1` matches label `RSeQC_3_0_1` for process with name post_mapping_QC:RSeQC
Mar-31 11:45:20.740 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: slurm
Mar-31 11:45:20.740 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'slurm'
Mar-31 11:45:20.842 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:Preseq_2_0_3` matches label `Preseq_2_0_3` for process with name post_mapping_QC:Lc_extrap
Mar-31 11:45:20.844 [main] DEBUG nextflow.executor.ExecutorFactory - << taskConfig executor: slurm
Mar-31 11:45:20.844 [main] DEBUG nextflow.executor.ExecutorFactory - >> processorType: 'slurm'
Mar-31 11:45:20.948 [main] DEBUG nextflow.script.ProcessConfig - Config settings `withLabel:subread_2_0_0_featureCounts` matches label `subread_2_0_0_featureCounts` for process with name featureCounts
Mar-31 11:45:21.042 [main] DEBUG nextflow.Session - Session aborted -- Cause: Process `featureCounts` declares 2 input channels but 3 were specified
Mar-31 11:45:21.351 [main] DEBUG nextflow.Session - The following nodes are still active:
  [operator] ifEmpty
  [operator] map
  [operator] ifEmpty
  [operator] ifEmpty
  [operator] ifEmpty
  [operator] ifEmpty
  [operator] map
  [operator] groupTuple
  [operator] map
  [operator] map
  [operator] collect
  [operator] map
  [operator] join
  [operator] map
  [operator] collect
  [operator] map
  [operator] collect
  [operator] collect

Mar-31 11:45:21.645 [main] ERROR nextflow.cli.Launcher - Process `featureCounts` declares 2 input channels but 3 were specified
nextflow.exception.ScriptRuntimeException: Process `featureCounts` declares 2 input channels but 3 were specified
	at nextflow.script.ProcessDef.run(ProcessDef.groovy:173)
	at nextflow.script.BindableDef.invoke_a(BindableDef.groovy:51)
	at nextflow.script.ComponentDef.invoke_o(ComponentDef.groovy:40)
	at nextflow.script.WorkflowBinding.invokeMethod(WorkflowBinding.groovy:87)
	at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeOnDelegationObjects(ClosureMetaClass.java:397)
	at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:339)
	at org.codehaus.groovy.runtime.callsite.PogoMetaClassSite.callCurrent(PogoMetaClassSite.java:64)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCallCurrent(CallSiteArray.java:51)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:156)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callCurrent(AbstractCallSite.java:184)
	at Script_e4262016$_runScript_closure1$_closure2.doCall(Script_e4262016:107)
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:497)
	at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:101)
	at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
	at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:263)
	at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1041)
	at groovy.lang.Closure.call(Closure.java:405)
	at groovy.lang.Closure.call(Closure.java:399)
	at nextflow.script.WorkflowDef.run0(WorkflowDef.groovy:204)
	at nextflow.script.WorkflowDef.run(WorkflowDef.groovy:191)
	at nextflow.script.BindableDef.invoke_a(BindableDef.groovy:51)
	at nextflow.script.ChainableDef$invoke_a.call(Unknown Source)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCall(CallSiteArray.java:47)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:115)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:127)
	at nextflow.script.BaseScript.runDsl2(BaseScript.groovy:180)
	at nextflow.script.BaseScript.run(BaseScript.groovy:189)
	at nextflow.script.ScriptParser.runScript(ScriptParser.groovy:225)
	at nextflow.script.ScriptRunner.run(ScriptRunner.groovy:218)
	at nextflow.script.ScriptRunner.execute(ScriptRunner.groovy:126)
	at nextflow.cli.CmdRun.run(CmdRun.groovy:273)
	at nextflow.cli.Launcher.run(Launcher.groovy:460)
	at nextflow.cli.Launcher.main(Launcher.groovy:642)
Mar-31 11:45:21.840 [Actor Thread 5] DEBUG nextflow.Nextflow - Ignore exit because execution is already aborted -- message=Bed12 file not found: /hpc/pmc_vanboxtel/data/STAR/Homo_sapiens.GRCh38/Homo_sapiens.GRCh38.76.bed12.sorted.bed
Mar-31 11:45:21.840 [Actor Thread 3] DEBUG nextflow.Nextflow - Ignore exit because execution is already aborted -- message=GTF file not found: /hpc/pmc_vanboxtel/data/homo_sapiens.GRCh38.GATK.illumina/Homo_sapiens.GRCh38.76.gtf
Mar-31 11:45:21.840 [Actor Thread 4] DEBUG nextflow.Nextflow - Ignore exit because execution is already aborted -- message=STAR index not found: /hpc/pmc_vanboxtel/data/STAR/Homo_sapiens.GRCh38
Mar-31 11:45:21.840 [Actor Thread 6] DEBUG nextflow.Nextflow - Ignore exit because execution is already aborted -- message=Transcripts fasta not found: /hpc/pmc_vanboxtel/data/STAR/Homo_sapiens.GRCh38/Homo_sapiens.GRCh38.76_transcripts_salmon/GRCh38_transcripts_salmon
Mar-31 11:45:21.844 [Actor Thread 2] ERROR nextflow.extension.OperatorEx - @unknown
nextflow.exception.ProcessUnrecoverableException: No R1 fastq.gz files found in /hpc/pmc_vanboxtel/projects/Markus_Karlijn_RNA/fastq/SRR1028343/!
	at sun.reflect.NativeConstructorAccessorImpl.newInstance0(Native Method)
	at sun.reflect.NativeConstructorAccessorImpl.newInstance(NativeConstructorAccessorImpl.java:62)
	at sun.reflect.DelegatingConstructorAccessorImpl.newInstance(DelegatingConstructorAccessorImpl.java:45)
	at java.lang.reflect.Constructor.newInstance(Constructor.java:422)
	at org.codehaus.groovy.reflection.CachedConstructor.invoke(CachedConstructor.java:80)
	at org.codehaus.groovy.runtime.callsite.ConstructorSite$ConstructorSiteNoUnwrapNoCoerce.callConstructor(ConstructorSite.java:105)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCallConstructor(CallSiteArray.java:59)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callConstructor(AbstractCallSite.java:237)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callConstructor(AbstractCallSite.java:249)
	at nextflow.Nextflow.error(Nextflow.groovy:261)
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:497)
	at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:101)
	at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
	at org.codehaus.groovy.runtime.callsite.StaticMetaMethodSite$StaticMetaMethodSiteNoUnwrap.invoke(StaticMetaMethodSite.java:131)
	at org.codehaus.groovy.runtime.callsite.StaticMetaMethodSite.callStatic(StaticMetaMethodSite.java:100)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCallStatic(CallSiteArray.java:55)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callStatic(AbstractCallSite.java:196)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.callStatic(AbstractCallSite.java:208)
	at Script_e11833b2$_extractAllFastqFromDir_closure7.doCall(Script_e11833b2:72)
	at Script_e11833b2$_extractAllFastqFromDir_closure7.doCall(Script_e11833b2)
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:497)
	at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:101)
	at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
	at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:263)
	at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1041)
	at org.codehaus.groovy.runtime.callsite.PogoMetaClassSite.call(PogoMetaClassSite.java:37)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCall(CallSiteArray.java:47)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:115)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:119)
	at nextflow.extension.OperatorEx$_ifEmpty_closure38.doCall(OperatorEx.groovy:1365)
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:497)
	at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:101)
	at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
	at org.codehaus.groovy.runtime.metaclass.ClosureMetaClass.invokeMethod(ClosureMetaClass.java:263)
	at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1041)
	at org.codehaus.groovy.runtime.callsite.PogoMetaClassSite.call(PogoMetaClassSite.java:37)
	at org.codehaus.groovy.runtime.callsite.CallSiteArray.defaultCall(CallSiteArray.java:47)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:115)
	at org.codehaus.groovy.runtime.callsite.AbstractCallSite.call(AbstractCallSite.java:127)
	at nextflow.extension.DataflowHelper$3.afterStop(DataflowHelper.groovy:258)
	at groovyx.gpars.dataflow.operator.DataflowProcessor.fireAfterStop(DataflowProcessor.java:324)
	at groovyx.gpars.dataflow.operator.DataflowProcessorActor.afterStop(DataflowProcessorActor.java:59)
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:497)
	at org.codehaus.groovy.reflection.CachedMethod.invoke(CachedMethod.java:101)
	at groovy.lang.MetaMethod.doMethodInvoke(MetaMethod.java:323)
	at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1217)
	at groovy.lang.MetaClassImpl.invokeMethod(MetaClassImpl.java:1041)
	at org.codehaus.groovy.runtime.InvokerHelper.invokePojoMethod(InvokerHelper.java:999)
	at org.codehaus.groovy.runtime.InvokerHelper.invokeMethod(InvokerHelper.java:990)
	at groovyx.gpars.actor.Actor.callDynamic(Actor.java:369)
	at groovyx.gpars.actor.Actor.handleTermination(Actor.java:320)
	at groovyx.gpars.actor.AbstractLoopingActor$1.handleMessage(AbstractLoopingActor.java:86)
	at groovyx.gpars.util.AsyncMessagingCore.run(AsyncMessagingCore.java:132)
	at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1142)
	at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:617)
	at java.lang.Thread.run(Thread.java:745)

FastQC report only included in MultiQC when TrimGalore step enabled

Disabling TrimGalore in the pipeline will still generate FastQC report for the raw data, however results are not included in final MultiQC report. When enabling TrimGalore the FastQC report for the trimmed data IS included.

*Fix
Add line
"fastqc_logs = FastQC.out.fastqc_report "

After FastQC(fastq_files) in first if statement in the pre_processing.nf workflow...

Generalize the test data

Test data is now hard-coded to local paths on the HPC.
Need to be added to the repository or made available for download.

Salmon quantmerge step is unnecessary when analysing a single sample and creates an error.

When running the pipeline with a single sample you get the following error when running the Salmon step.

Execution completed unsuccessfully!

The exit status of the task that caused the workflow execution to fail was: 0.

The full error message was:

Error executing process > 'alignment_free_quant:QuantMerge (Salmon QuantMerge pre_NFpipeline)'

Caused by:
Missing output file(s) *.txt expected by process alignment_free_quant:QuantMerge (Salmon QuantMerge pre_NFpipeline)

Command executed:

salmon quantmerge -c numreads --quants {Breast} -o pre_NFpipeline_transcripts_quantmerge_numReads.txt
salmon quantmerge -c tpm --quants {Breast} -o pre_NFpipeline_transcripts_quantmerge_TPM.txt
salmon quantmerge -c len --quants {Breast} -o pre_NFpipeline_transcripts_quantmerge_Length.txt
salmon quantmerge -c elen --quants {Breast} -o pre_NFpipeline_transcripts_quantmerge_EffectiveLength.txt

Command exit status:
0

Command output:
[2021-03-03 00:38:17.817] [mergeLog] [info] samples: [ {Breast} ]
[2021-03-03 00:38:17.817] [mergeLog] [info] sample names : [ {Breast} ]
[2021-03-03 00:38:17.817] [mergeLog] [info] output column : TPM
[2021-03-03 00:38:17.817] [mergeLog] [info] output file : pre_NFpipeline_transcripts_quantmerge_numReads.txt
[2021-03-03 00:38:17.817] [mergeLog] [critical] The sample directory {Breast} either doesn't exist, or doesn't contain a quant.sf file
[2021-03-03 00:38:17.864] [mergeLog] [info] samples: [ {Breast} ]
[2021-03-03 00:38:17.864] [mergeLog] [info] sample names : [ {Breast} ]
[2021-03-03 00:38:17.864] [mergeLog] [info] output column : TPM
[2021-03-03 00:38:17.864] [mergeLog] [info] output file : pre_NFpipeline_transcripts_quantmerge_TPM.txt
[2021-03-03 00:38:17.864] [mergeLog] [critical] The sample directory {Breast} either doesn't exist, or doesn't contain a quant.sf file
[2021-03-03 00:38:17.891] [mergeLog] [info] samples: [ {Breast} ]
[2021-03-03 00:38:17.891] [mergeLog] [info] sample names : [ {Breast} ]
[2021-03-03 00:38:17.891] [mergeLog] [info] output column : TPM
[2021-03-03 00:38:17.891] [mergeLog] [info] output file : pre_NFpipeline_transcripts_quantmerge_Length.txt
[2021-03-03 00:38:17.891] [mergeLog] [critical] The sample directory {Breast} either doesn't exist, or doesn't contain a quant.sf file
[2021-03-03 00:38:17.917] [mergeLog] [info] samples: [ {Breast} ]
[2021-03-03 00:38:17.917] [mergeLog] [info] sample names : [ {Breast} ]
[2021-03-03 00:38:17.917] [mergeLog] [info] output column : TPM
[2021-03-03 00:38:17.917] [mergeLog] [info] output file : pre_NFpipeline_transcripts_quantmerge_EffectiveLength.txt
[2021-03-03 00:38:17.917] [mergeLog] [critical] The sample directory {Breast} either doesn't exist, or doesn't contain a quant.sf file

The problem is that there are no outputs from the QuantMerge step in https://github.com/UMCUGenetics/RNASeq-NF/blob/master/sub-workflows/alignment_free_quant.nf
This step is not needed for a single sample because there is only 1 output to merge.

A solution would be to check how many samples are being analysed. If it is only 1 sample, skip the QuantMerge step and set the single quant.sf file as output.

Missing modules: Expression quantification

Merge HTSeq counts
RPKM normalization

Error executing process > 'pre_processing:FastQC

I try to run the pipeline but it gives me an error:

Pulling Singularity image docker://quay.io/biocontainers/trim-galore:0.6.5--0 [cache /hpc/local/CentOS7/pmc_vanboxtel/singularity_cache/quay.io-biocontainers-trim-galore-0.6.5--0.img]
Pulling Singularity image docker://quay.io/biocontainers/fastqc:0.11.8--1 [cache /hpc/local/CentOS7/pmc_vanboxtel/singularity_cache/quay.io-biocontainers-fastqc-0.11.8--1.img]
Error executing process > 'pre_processing:FastQC (FastQC SRR1028343 - SRR1028343_C0PNNACXX_3)'

Caused by:
  Failed to pull singularity image
  command: singularity pull  --name quay.io-biocontainers-fastqc-0.11.8--1.img docker://quay.io/biocontainers/fastqc:0.11.8--1 > /dev/null
  status : 255
  message:
    time="2020-06-02T09:20:07+02:00" level=warning msg="\"/run/user/60165\" directory set by $XDG_RUNTIME_DIR does not exist. Either create the directory or unset $XDG_RUNTIME_DIR.: stat /run/user/60165: no such file or directory: Trying to pull image in the event that it is a public image."
    INFO:    Converting OCI blobs to SIF format
    INFO:    Starting build...
    time="2020-06-02T09:20:08+02:00" level=warning msg="\"/run/user/60165\" directory set by $XDG_RUNTIME_DIR does not exist. Either create the directory or unset $XDG_RUNTIME_DIR.: stat /run/user/60165: no such file or directory: Trying to pull image in the event that it is a public image."
    time="2020-06-02T09:20:09+02:00" level=warning msg="\"/run/user/60165\" directory set by $XDG_RUNTIME_DIR does not exist. Either create the directory or unset $XDG_RUNTIME_DIR.: stat /run/user/60165: no such file or directory: Trying to pull image in the event that it is a public image."
    Getting image source signatures
    Copying blob sha256:a3ed95caeb02ffe68cdd9fd84406680ae93d633cb16422d00e8a7c22955b46d4
    Copying blob sha256:77c6c00e8b61bb628567c060b85690b0b0561bb37d8ad3f3792877bddcfe2500
    Copying blob sha256:3aaade50789a6510c60e536f5e75fe8b8fc84801620e575cb0435e2654ffd7f6
    Copying blob sha256:00cf8b9f3d2a08745635830064530c931d16f549d031013a9b7c6535e7107b88
    Copying blob sha256:7ff999a2256f84141f17d07d26539acea8a4d9c149fefbbcc9a8b4d15ea32de7
    Copying blob sha256:d2ba336f2e4458a9223203bf17cc88d77e3006d9cbf4f0b24a1618d0a5b82053
    Copying blob sha256:dfda3e01f2b637b7b89adb401f2f763d592fcedd2937240e2eb3286fabce55f0
    Copying blob sha256:a3ed95caeb02ffe68cdd9fd84406680ae93d633cb16422d00e8a7c22955b46d4
    Copying blob sha256:a3ed95caeb02ffe68cdd9fd84406680ae93d633cb16422d00e8a7c22955b46d4
    Copying blob sha256:10c3bb32200bdb5006b484c59b5f0c71b4dbab611d33fca816cd44f9f5ce9e3c
    Copying blob sha256:a3ed95caeb02ffe68cdd9fd84406680ae93d633cb16422d00e8a7c22955b46d4
    Copying blob sha256:5bb2f56dde285963c99dc3fb9a11d1e024d08aa1be97d31981524cdcc643a62a
    Copying config sha256:84810ed56e9f1a139355a9df39aa893f7530e5ecd3580b857102422629556c4b
    Writing manifest to image destination
    Storing signatures
    FATAL:   While making image from oci registry: while building SIF from layers: conveyor failed to get: no descriptor found for reference "0745999ecde65d2a1ee1d4ed10fc543e71feb7dc7a4079bbb1a43c71c0c73508"

Any suggestions/ideas to solve this problem?

Star genomeGenerate
GTF /GFFto BED12 conversion (RSeQC)

Missing modules: RNASeq variant calling

We will implement the GATK4.0 BP workflow and skip the old recommendations for GATK 3.0.

MergeBamAlignment (Picard)
FastqToSam (Picard)
SplitNCigarReads (GATK 4.0)
Apply Recalibration (GATK 4.0)
AnalyzeCovariates (GATK 4.0)

Additional modules: Expression quantification

Salmon (quant & index)
RSEM (rsem-calculate-expression, rsem-prepare-reference )

ReadAlignChunk_processChunks.cpp:161:processChunks EXITING because of FATAL ERROR in input reads: unknown file format: the read ID should start with @ or >

Hi TIlman

I think If you want to use compressed fastq file as input you need to use the option --readsFilesCommand zcat|gunzip -c in stead of --readFilesIn.

See the STAR manual 3.1.

Option: skip analysis

Implement option to skip one or multiple steps of the workflow.

Refactoring

Move each tool to a sub workflow

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