miARma/bin/Linux/cutadapt/cutadapt/calign.so: undefined symbol: PyString_Type

[Zhenyu li]

Hi, I'm tring to use miARmaSeq to analyse miRNA data. I downloaded miARma_pipelineV1.5.tar.bz2 and the test dataset from https://sourceforge.net/projects/miarma/files/Examples/Examples_miARma_miRNAs.tar.bz2. I used the commandline below. ./miARma Examples/basic_examples/miRNAs/Known_miRNAs/Known_miRNAs_pipeline.ini. However, I ran into an error. The output was:

miARma, miRNA and RNASeq Multiprocess Analysis

miARma v 1.5 (Apr-2016)

Created at Computational Biology and Bioinformatics Group (CbBio)

Institute of Biomedicine of Seville. IBIS (Spain)

Copyright (c) 2016 IBIS. All rights reserved.

mail : [e-mail]

[Thu Apr 20 14:28:41 2017] Starting a miARma analysis for miRNA [Thu Apr 20 14:28:41 2017] Checking provided parameters for: Quality,Adapter,Aligner,ReadCount,DEAnalysis,TargetPrediction. [Thu Apr 20 14:28:41 2017] All parameters are correct. [Thu Apr 20 14:28:41 2017] Starting Quality Analysis. [Thu Apr 20 14:29:35 2017] Quality Analysis finished. [Thu Apr 20 14:29:35 2017] Checking if files in folder: "Examples/basic_examples/miRNAs/reads/" are in the correct fastq format. [Thu Apr 20 14:29:36 2017] Starting a Adapter removal analysis CUTADAPT ERROR :: system args failed: 256 (mkdir -p Examples/basic_examples/miRNAs/Known_miRNAs/results///cutadapt_results/ ; cut_adapt -b ATCTCGTATGCCGTCTTCTGCTTGAA -m 15 -M 35 -q 0 Examples/basic_examples/miRNAs/reads///SRR873382.fastq.gz > Examples/basic_examples/miRNAs/Known_miRNAs/results///cutadapt_results/SRR873382_cut.fastq 2>> Examples/basic_examples/miRNAs/Known_miRNAs/results//miARma_stat.31658.log) at lib/CbBio/RNASeq/Adapt.pm line 851.

And the last 10 lines of file 'Examples/basic_examples/miRNAs/Known_miRNAs/results//miARma_stat.31658.log' are: Traceback (most recent call last): File "./bin/Linux/cutadapt//cut_adapt", line 9, in <module> from cutadapt.scripts import cutadapt File "/data/bin/miARma/bin/Linux/cutadapt/cutadapt/scripts/cutadapt.py", line 74, in <module> from cutadapt.adapters import Adapter, ColorspaceAdapter, BACK, FRONT, PREFIX, ANYWHERE File "/data/bin/miARma/bin/Linux/cutadapt/cutadapt/adapters.py", line 4, in <module> from cutadapt import align, colorspace File "/data/bin/miARma/bin/Linux/cutadapt/cutadapt/align.py", line 225, in <module> from cutadapt.calign import globalalign_locate ImportError: /data/bin/miARma/bin/Linux/cutadapt/cutadapt/calign.so: undefined symbol: PyString_Type

I googled it but got nothing helpful. Could you please help? Thanks in advance.

[Eduardo Andres Leon]

Dear Zhenyu Li, First of all, I'd like to thank you for using miARma.

Then, I start by recommend you the latest version of miARma, which is 1.6.1 and is accesible at : https://bitbucket.org/cbbio/miarma/get/master.tar.bz2

But your problem seems to be related with python. Which python version are your using ? And which OS ?

Regards

[Zhenyu li]

Thank you for quick response.

The python version is 3.5.2. And the OS is 'Linux VM-243-225-ubuntu 3.13.0-36-generic x86_64 GNU/Linux'.

I will also try the latest version you provided.

[Eduardo Andres Leon]

In my machine I’ve installed python 2.7.12 and 3.5.2. If I use python3, cut adapt doesn’t work. So I guess is mandatory to use python 2.7 to make this software works

python2.7 ~/bin/miARma/bin/Linux/cutadapt/cut_adapt --version
1.3
python3.5 ~/bin/miARma/bin/Linux/cutadapt/cut_adapt --version
Traceback (most recent call last):
  File "/home/eandres/bin/miARma/bin/Linux/cutadapt/cut_adapt", line 9, in <module>
    from cutadapt.scripts import cutadapt
  File "/home/eandres/bin/miARma1.6.1/bin/Linux/cutadapt/cutadapt/scripts/cutadapt.py", line 74, in <module>
    from cutadapt.adapters import Adapter, ColorspaceAdapter, BACK, FRONT, PREFIX, ANYWHERE
  File "/home/eandres/bin/miARma1.6.1/bin/Linux/cutadapt/cutadapt/adapters.py", line 4, in <module>
    from cutadapt import align, colorspace
  File "/home/eandres/bin/miARma1.6.1/bin/Linux/cutadapt/cutadapt/align.py", line 225, in <module>
    from cutadapt.calign import globalalign_locate
ImportError: /home/eandres/bin/miARma1.6.1/bin/Linux/cutadapt/cutadapt/calign.so: undefined symbol: PyString_Type

I’ve seen that the last cut adapt version also supports python 3.5, so I will update it in the coming months

Meanwhile, if you are using Ubuntu, yo can install python2.7: apt-get install python2.7

[Zhenyu li]

Yes, you are right. I used python 2.7 and the error didn't happen. However, I ran into another error. The related output is

"[Fri Apr 21 12:13:18 2017] Starting a differential expression analysis using EdgeR software(s)

Problem while running this R command:

print(resultsfiles)

Error:

print(resultsfiles) : object 'resultsfiles' not found

Execution halted"

And the output in log file is

QC_EdgeR :: [Fri Apr 21 12:13:19 2017] Executing                setwd("/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results/Readcount_results")
                source("http://valkyrie.us.es/CbBio/RNASeq/R-Scripts/QC_EdgeR.R")
                resultsfiles<-QC_EdgeR(projectdir="/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results",dir="/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results/Readcount_results", file="cut_bw1-ReadCount.tab", targetfile="/data/bin/miARma/Examples/basic_examples/miRNAs/data/targets.txt", label="Hypoxia_cut_bw1", filter="yes")

This happened for both v1.5 and the latest version. The R version I used is 3.3.1 (2016-06-21).

[Eduardo Andres Leon]

Hi, Don’t worry, this is the most frequent error.

You should check that bioconductor is properly installed, so I recommend you to enter in a R console and execute:

source("https://bioconductor.org/biocLite.R”)

biocLite(“edgeR")

In once of these two steps, bioconductor has to ask for a personal library to install the software, you have to say yes. This is important because miARma needs to install several packages.

Let me know

[Eduardo Andres Leon]

And by the way (in case everything is properly installed), you can open a R console and execute (for miARma 1.6.1):

R>setwd("/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results/Readcount_results”)

R>source("/data/bin/miARma/lib/CbBio/RNASeq/R-Scripts/QC_EdgeR.R”) # you should check that I wrote correctly this path

R>QC_EdgeR(projectdir="/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results",dir="/data/bin/miARma/Examples/basic_examples/miRNAs/Known_miRNAs/results/Readcount_results", file="cut_bw1-ReadCount.tab", targetfile="/data/bin/miARma/Examples/basic_examples/miRNAs/data/targets.txt", label="Hypoxia_cut_bw1", filter="yes”)

This will also tell you what problem are you experiencing

Regards

[Zhenyu li]

After intalling edgeR and other requried packages, the pipeline is working now! It took about 10 min to run from the first step to differential expression analysis. But the last step 'target prediction analysis' seems to need very long time. It hasn't finished after running more than one hour. Is this normal?

[Eduardo Andres Leon]

it could be. The target prediction is the most computational expensive part. In order to see if everything is ok, you can access to the target prediction folder to see how the file is changing

Edu