Problem with running Differential expression & Functional analysis modules in miARma 1.7

[Mahdee]

Hi, Im experiencing a problem while running the differential expression analysis. I installed miARma1.7 on Ubuntu 16.04 LTS. I exactly followed the guidelines for miARma installations, But when I want to implement differential expression analysis, i see the following error:

Executing source("./lib/CbBio/RNASeq/R-Scripts/QC_EdgeR.R") setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results") resultsfiles<-QC_EdgeR(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b",dir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results", file="his-ReadCount.tab", targetfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/targets.txt", label="Meth_his", filter="yes") DE_EDGER :: [Sun 18:54:01 2018] Starting Differential Expression Analysis of /home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results/his-ReadCount.tab with EdgeR DE_EDGER :: [Sun 18:54:01 2018] Executing source("./lib/CbBio/RNASeq/R-Scripts/DE_EdgeR.R") setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results") resultsfiles<-DE_EdgeR(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/EdgeR_results",dir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results", file="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results/his-ReadCount.tab", targetfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/targets.txt", label="Meth_his", contrastfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/contrast.txt", filter="yes", replicates="no", bcvvalue=0.3) Problem while running this R command: print(resultsfiles) Error: print(resultsfiles) : object 'resultsfiles' not found Execution halted

I would be grateful, if you could help me.

[Eduardo Andres Leon]

Hi, Could you try this in your terminal?:

bash$>cd /home/mahnet/NGS/miARma1.7.0/
bash$>R
R> source("./lib/CbBio/RNASeq/R-Scripts/QC_EdgeR.R")
R>setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results")
R>QC_EdgeR(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b",dir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results", file="his-ReadCount.tab", targetfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/targets.txt", label="Meth_his", filter="yes")

This should print the error

[Mahdee]

Thanks for your reply, I ran following commands as you mentioned and the result was as follows below Loading required package: edgeR Loading required package: limma Loading required package: ggplot2 Loading required package: gplots Attaching package: ‘gplots’ The following object is masked from ‘package:stats’: lowess Loading required package: genefilter 12:01:43 Starting quality control analysis of Meth_his 12:01:44 The file Meth_his_QC_Report.pdf has been generated in /home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b [1] "/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Meth_his_QC_Report.pdf"

the link below shows the generated PDF file Link Text

[Eduardo Andres Leon]

Fine, so we move to the next step:

bash$>cd /home/mahnet/NGS/miARma1.7.0/
bash$>R
R> source("./lib/CbBio/RNASeq/R-Scripts/DE_EdgeR.R") 
R>setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results")
R> DE_EdgeR(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/EdgeR_results",dir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results", file="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results/his-ReadCount.tab", targetfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/targets.txt", label="Meth_his", contrastfile="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/data/contrast.txt", filter="yes", replicates="no", bcvvalue=0.3)

[Mahdee]

Dear Eduardo, I got this message Loading required package: edgeR Loading required package: limma 14:08:37 Starting Differential expression analysis with EdgeR of Meth_his Error in exactTest(dgenorm, pair = c(contrastval[2], contrastval[1]), : At least one element of given pair is not a group. Groups are: Treated

I could resolve this issue by changing the targets.txt file. Now it works!! by the way, inside targets.txt file, the elements were separated by space or by tab??

[Eduardo Andres Leon]

Cool!

targets.txt elements should be always separated by tab

[Mahdee]

Thank you for all your help!

[Mahdee]

Dear Eduardo, after successfully finishing up differential expression analysis, when i ran functional analysis, the below error showed up: Problem while running this R command: print(resultsfiles) Error: print(resultsfiles) : object 'resultsfiles' not found Execution halted

I implemented the following commands in R to examine the issue: source("./lib/CbBio/RNASeq/R-Scripts/F_Analysis.R") setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results") resultsfiles<-NA resultsfiles<-F_Analysis(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results",up="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.up_entities_edgeR.txt",down="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.down_entities_edgeR.txt",universe="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.universe_entities_edgeR.txt",organism="rat",method="edgeR",seq_id="transcript_id",label="Meth_his_Comp")

I came up with this error after the examination: Error in file(filename, "r", encoding = encoding) : cannot open the connection to 'http://bioconductor.org/biocLite.R' In addition: Warning message: In file(filename, "r", encoding = encoding) : URL 'http://bioconductor.org/biocLite.R': status was 'Failure when receiving data from the peer'

Could you please tell me what is going on ? Thanks in advance

[Eduardo Andres Leon]

Hi Mahdee,

As we did before, can you try:

bash$>cd /home/mahnet/NGS/miARma1.7.0/
bash$>R
R> source("./lib/CbBio/RNASeq/R-Scripts/F_Analysis.R") 
R> setwd("/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Readcount_results")
R> F_Analysis(projectdir="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results",up="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.up_entities_edgeR.txt",down="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.down_entities_edgeR.txt",universe="/home/mahnet/NGS/miARma1.7.0/Examples/basic_examples/mRNAs/b/Functional_Analysis_results/.universe_entities_edgeR.txt",organism="rat",method="edgeR",seq_id="transcript_id",label="Meth_his_Comp")

[Mahdee]

Hi, the output is as follows below,

Error in file(filename, "r", encoding = encoding) : cannot open the connection to 'http://bioconductor.org/biocLite.R' In addition: Warning message: In file(filename, "r", encoding = encoding) : URL 'http://bioconductor.org/biocLite.R': status was 'Failure when receiving data from the peer'

[Eduardo Andres Leon]

Are you using the correct seq_id parameter in de FA_Analysis section ?

Check that the 3 files exists: up/down and universe file. The full path is in the last command you write in my previous post

[Mahdee]

Yes, I used the same "transcript_id" as seqid parameter in my Read count analysis. Moreover, there was no problem with this parameter in read count analysis.

[Mahdee]

Yes. all the three files exist, however, down.txt file is empty

[Mahdee]

Dear Eduardo,

Is there any more suggestions?

[Eduardo Andres Leon]

Hi, According with the summary (xls) file, did you have down/up DEG ?

[Mahdee]

There is no xls file related to functional analysis, but as you see in the attached file, the there is a section related to NO. of DEG

[Eduardo Andres Leon]

Yes, but the number of DEG is really small (FA_Analysis uses by default FDR<=0.05), as a test, could you include a greater threshold in the FA_Analysis?: edger_cutoff=0.15 .... edger_cutoff=0.5

Remember that you can comment all sections except FA_Analysis in order to rerun just this analysis

[Mahdee]

Dear Eduardo, I tried various edger_cutoff values as you suggested, but still the same error comes up:

Problem while running this R command: print(resultsfiles) Error: print(resultsfiles) : object 'resultsfiles' not found Execution halted

[Eduardo Andres Leon]

What about the files ? are still empty ?

[Mahdee]

There is no xls file and all the three txt files exist, however, down.txt file is empty

[Eduardo Andres Leon]

Did you see if you already have downregualted genes y the EdgeR_Results folder (in the xls result file)?

[Mahdee]

Yes, there is down-regulated genes in the xls file inside EdgeR_results folder

[Eduardo Andres Leon]

having a FDR<0.05 and a log2FC<0 ?

[Mahdee]

Yes exactly,

[Eduardo Andres Leon]

Could it be possible to get your data ? (of course this will be just for testing issues).

If possible, I'll tell you what folders and files I'll need, so you can send me by mail/weTransfer

[Mahdee]

Okay, No problem,

It would be better to me to email you rather than exposing them to this forum.

[Eduardo Andres Leon]

Perfect

my email is eduardo.andres at csic.es

And I'll need:

• ini file
• EdgeR_Results folder
• FAnalisis_Results folder
• target.txt
• contrast.txt

This should be enough Thanks !