rf-map&rf-count

[Shuang1248]

Hi Danny,

   I am using RNA framework to analyze  my SHAPE-seq data, but I got the similar error when I used rf-map and rf-count like this:
   Can't use an undefined value as an ARRAY reference at RNAFramework-master/rf-map line 444.
   Can't use an undefined value as an ARRAY reference at RNAFramework-master/rf-count line 619.
   These have been bothering me for a long time，could you please help me?

Thanks

[dincarnato]

Hi,

To understand what is going on I need the files you are working with (or a small case example to reproduce the issue) + the exact command line you are executing. Also, can you run rf-map with the -kl option and share the logs/ folder in the output?

Danny

[Shuang1248]

Hi Danny, This is the exact command line I am executing: rf-map -b2 -p 20 -o rf_map -ca3 AGATCGGAAGAGC -cl 18 -ctn -b5 3 -bi /data/RNAframework/transcriptome/transcriptome_sorted -bnr SRR933552.fastq.gz -kl This is the log file: SRR933552_cutadapt.log And I sent the other files to the email dincarnato@rnaframework.com.

Thanks for your help!

[dincarnato]

Is that the only log file you have? Are you sure bowtie2 is installed on your computer and in PATH? I didn't receive anything to my email. Please upload the files somewhere.

[Shuang1248]

Hi Dannny, I am not sure why you can not receive my email, to solve this problem about rf_map, I try to trim adapters by cutadapt and map reads by bowtie2 and sort by samtools instead of using rf_map, and then I use rf_count to calculate the RT stops, can I analyze by this way? However, when I use rf_count, I also got an error: Can't use an undefined value as an ARRAY reference at RNAFramework-master/rf-count line 619. And this is my exact command line: rf-count -p 4 -o rf_count -f ../transcriptome/transcriptome.sorted.fa -ndd --sorted Map.sorted.bam Because you can not receive my email, so I attach some pictures of files I used.

Thanks

[dincarnato]

Sorry Shuang but I cannot reproduce the issue. I am gonna need the files. Please upload them somewhere (drive, wetransfer, etc.).

[Shuang1248]

Hi Danny, I upload data on wetransfer, this is the link: https://we.tl/t-rg47Mfy9jY , I hope this link will work. My command line: rf-count -r -f ~/transcriptome/transcriptome.sorted.fa Map.sorted.bam The information of error: Can't use an undefined value as an ARRAY reference at RNAFramework-master/rf-count line 619.

Thanks!

[dincarnato]

Hi Shuang,

sorry but it looks there is an issue with your RNAFramework installation. The files get processed smoothly on my system.

$ rf-count -r -f transcriptome.sorted.fa Map.sorted.bam

[+] Making output directory...
[+] Guessing file types:

  Sample        Type    5'-end trimming

  Map.sorted    BAM     0 nt

[+] Getting transcripts from reference, and building count table base structure...
[+] Inspecting SAM/BAM file headers...
[+] Assuming that provided SAM/BAM files are already sorted. Skipping sorting...
[+] Calculating per-base RT-stops and coverage. This may take a while...

  [-] Processing sample "Map.sorted" (PID: 2757)

Please delete your local install and re-clone the RNAFramework from this repository. Also make sure you have a recent version of Perl installed on your system (5.22 or greater).

Best, Danny

[Shuang1248]

Hi Danny, I can get these outputs as well ,but after " [-] Processing sample "Map.sorted" (PID: 2757)", it will inform me of the error: Can't use an undefined value as an ARRAY reference at RNAFramework-master/rf-count line 619. If you didn't get the error, I think it may because there is an issue with my RNAFramework installation.

Thanks

[dincarnato]

Ok, then let me finish running the analysis and see if I get the error as well. This is why I asked for the FULL OUTPUT.

[dincarnato]

It just finished running smoothly:

[+] Statistics:

  [*] Sample "Map.sorted": 16553 transcripts covered [A: 22.81; C: 30.32; G: 18.66; T: 28.21]

[+] Cleaning up temporary files...
[+] All done.

Please make sure you have the latest version of the RNA Framework (git pull) and a recent version of SAMTools.

[Shuang1248]

Hi, Danny: Thank you very much, I updated the samtools, and I can run smoothly successfully!

[dincarnato]

Great! Best, Danny