Bowtie 2 for circular RNAs

Question

[neha 114]

Hello,

I am working with bowtie 2 tool and new to this field thus trying to reproduce the work of Memczak, Sebastian, et al. "Circular RNAs are a large class of animal RNAs with regulatory potency." Nature 495.7441 (2013): 333-338.

Can anyone please suggest me how to use the circ.py script described by them.

I have tried it but getting no results in .bed and .reads file.

Kindly suggest, I will be highly thankful.

[GouthamAtla]

Can you point out where is the script circ.py?

[neha 114]

Thank you for the reply.

The scripts used are available at http://www.circbase.org/

[GouthamAtla]

Hi, there is a README in the software package with detailed instructions how to run the program. You need to follow that and evaluate the output at each step.

Then you may figure out at what step the things are going wrong. If you feel everything is fine but the problem is only with the find_circ.py script, share the input file used for that script with exact command. Its really difficult to tell what went wrong without looking at input files, if a software fail silently.

[neha 114]

I am obtaining this warning message while commands run:

Warning: -M is deprecated. Use -D and -R to adjust effort instead.
Warning: Could not open read file "ce6_anchors.qfa" for reading; skipping...
Error: No input read files were valid
bowtie2-align exited with value 1

[GouthamAtla]

That indicates the file ce6_anchors.qfa did not get created. From the README, these are the steps to create the file.

bowtie2 -p16 --very-sensitive --phred64 --mm -M20 --score-min=C,-15,0 -x bt2_ce6 -q -U <your_reads.qfa.gz> 2> bowtie2.log | samtools view -hbuS - | samtools sort - test_vs_ce6
samtools view -hf 4 test_vs_ce6.bam | samtools view -Sb - > unmapped_ce6.bam ./unmapped2anchors.py unmapped_ce6.bam | gzip > ce6_anchors.qfa.gz

So you should figure out in which step it went wrong. But Ideally it should say could not find ce6_anchors.qfa.gz are you giving ce6_anchors.qfa or ce6_anchors.qfa.gz?

[neha 114]

Hello,

Goutham really thanks for the reply, but will you please have a look at my directory from where I am running this command.

Kindly help please.

neha@neha-HP-Z820-Workstation:~$ cd memczek_et_al_data/
neha@neha-HP-Z820-Workstation:~/memczek_et_al_data$ ls

bt2_ce6.1.bt2 bt2_ce6.rev.2.bt2 find_circ.py test_vs_ce6.bam
bt2_ce6.2.bt2 ce6_anchors.qfa genome test_vs_ce6.sam
bt2_ce6.3.bt2 ce6_anchors.qfa.gz run_folder unmapped2anchors.py
bt2_ce6.4.bt2 ce6.fa SRR2104398_1.fastq unmapped_ce6.bam
bt2_ce6.rev.1.bt2 find_circ SRR2104398_2.fastq

neha@neha-HP-Z820-Workstation:~/memczek_et_al_data$ cd genome
neha@neha-HP-Z820-Workstation:~/memczek_et_al_data/genome$ ls

chrI.fa chrII.fa chrIII.fa chrIV.fa chrM.fa chrV.fa chrX.fa

neha@neha-HP-Z820-Workstation:~/memczek_et_al_data/genome$ cd ..
neha@neha-HP-Z820-Workstation:~/memczek_et_al_data$

I have successfully completed the previous steps , and now using the circ.py script (as mentioned in the readme) using the following command:

bowtie2 -p 16 --reorder --mm -M20 --score-min=C,-15,0 -q -x bt2_ce6 -U ce6_anchors.qfa

After this,

./find_circ.py -G genome -p ce6_test_ -s run_folder/sites.log > run_folder/sites.bed 2> run_folder/sites.reads

[AndreiR]

any errors?

[neha 114]

Hello,

After running the command,I am getting the log file having all the statistical parameters, but the .bed file and .reads file obtained are empty. kindly help.

Answer 1

[Nicolas Rosewick]

You could use STAR to detect circular RNAs from RNA-Seq: https://groups.google.com/forum/?hl=fr#!searchin/rna-star/circular/rna-star/Tmffpp4mPzc/jFhvaW5F1FIJ