Facing problem while performing circRNA Prediction with CIRI_AS

Question

[abdul.suboor123]

I am predicting circRNA in my datasets, while running this command

perl CIRI_AS_v1.2.pl -S Splitmap.sam -C CIRI2out.ciri -F Zea_mays.AGPv4.dna.chromosome.1.fa -A annotation.gtf -O CIRI2ASout -D yes

So the following error come after entering the above command.

Please make sure the SAM is the same one used for circRNA detection by CIRI without modification!

What could be the problem? even I have putted right information.

[GenoMax]

Facing problem while using perl command

Please use a more informative title. Adding that you are getting an error with ciri program in the title would make it more relevant.

[Kevin Blighe]

The answer is given on the SourceForge page: https://sourceforge.net/projects/ciri/files/CIRI-AS/

Q&A: Q: An error "Please make sure the SAM is the same one used for circRNA detection by CIRI without modification!" when running CIRI-AS after CIRI2? A: Because CIRI-AS was developed before CIRI2, it cannot process sequencing reads with different lengths whereas CIRI2 can. It seems that your sequencing reads were trimmed to different lengths before mapping, so the output of CIRI2 based on these reads cannot be recognized by CIRI-AS. My suggestion is to use raw reads directly, or trim all reads to the same length before BWA, CIRI2 and CIRI-AS.

So, the options:

• just align your raw reads using BWA mem without any trimming
• trim your raw reads to the same length (e.g. 70bp) prior to using BWA mem

Kevin

[abdul.suboor123]

Thank You @Kevin Bighe, I am following this procedure now...

Answer 1

[Kevin Blighe]

Here is a full working version of CIRI-full v2.0 using

• BWA stored at /Programs/bwa-0.7.12/
• CIRI-full v2.0 downloaded from https://sourceforge.net/projects/ciri/files/CIRI-full/
• Ubuntu 16.04
• java version "1.8.0_161" (Java(TM) SE Runtime Environment (build 1.8.0_161-b12); Java HotSpot(TM) 64-Bit Server VM (build 25.161-b12, mixed mode))
• Perl 5 version 22, subversion 1 (v5.22.1) (built for x86_64-linux-gnu-thread-multi (with 69 registered patches, see perl -V for more detail))

NOTE: Input test reads have the same length.

NOTE: the paths to CIRI programs in the program manual are incorrect

1

cd CIRI-full_v2.0/CIRI-full_test/

2

mkdir test_output

3

/Programs/bwa-0.7.12/bwa index test_ref.fa

...

4

/Programs/bwa-0.7.12/bwa mem -T 19 test_ref.fa test_1.fq.gz test_2.fq.gz > test_output/test.sam

...

5

perl ../bin/CIRI_v2.0.6/CIRI2.pl -I test_output/test.sam -O test_output/test.ciri -F test_ref.fa -A test_anno.gtf

...
 Candidate reads with splicing signals: 2994
 Candidate reads with PEM signals: 2994
 Candidate circRNAs found: 88
...
Number of circular RNAs found: 88
[Sun Aug 19 11:03:13 2018] CIRI finished its work. Please see output file test_output/test.ciri for detail.

6

perl ../bin/CIRI_AS_v1.2/CIRI_AS_v1.2.pl -S test_output/test.sam -C test_output/test.ciri -F test_ref.fa -A test_anno.gtf -O test_output/test -D yes

...
Loading circRNAs from test_output/test.ciri...
88 circRNAs are loaded.
Clustering circRNAs according to their locations...
[------------------------------------------------->] 100.0 %
88 circRNAs are divided into 22 clusters.
Scanning SAM file for splicing detection within circRNAs...
[------------------------------------------------->] 100.0 %
5493 candidate splice junctions within circRNAs from 2954 reads are recognized, of which 5493 are from known BSJ reads.
Splicing signals checking starts...
[------------------------------------------------->] 100.0 %
5461 candidate splice junctions have splicing signals.
Clustering starts...
In sum, 139 non-redundant splice junctions within circRNAs are detected.
Cirexon prediction according to splice junctions and sequencing depths...
[------------------------------------------------->] 100.0 %
Time Usage:
...

7

java -jar ../CIRI-full.jar RO1 -1 test_1.fq.gz -2 test_2.fq.gz -o test_output/test

...

8

/Programs/bwa-0.7.12/bwa mem -T 19 test_ref.fa test_output/test_ro1.fq > test_output/test_ro1.sam

...

9

java -jar ../CIRI-full.jar RO2 -r test_ref.fa -s test_output/test_ro1.sam -l 250 -o test_output/test

...
Full_length merged RO reads number= 86
Only contain 5'RO merged RO reads number= 49
see detail in file: /home/kblighe/Escritorio/CIRI-full_v2.0/CIRI-full_test/test_output/test_ro2_info.list

10

java -jar ../CIRI-full.jar Merge -c test_output/test.ciri -as test_output/test_jav.list -ro test_output/test_ro2_info.list -a test_anno.gtf -r test_ref.fa -o test_output/test

...
Loading Annotation...
Annotation Loaded
Loading CIRI_AS output...
CIRI_AS output Loaded
Loading CIRI output...
CIRI output Loaded
Loading CIRI_RO output...
CIRI_RO output Loaded
Combine AS and RO output
Combine completed, 135 reads are used
Outputing detail annotation file : /home/kblighe/Escritorio/CIRI-full_v2.0/CIRI-full_test/test_output/test_merge_circRNA_detail.anno
Merge complete. Start reconstruction.
Time: 0s

11

unset DISPLAY

12

java -jar ../CIRI-vis.jar -i test_output/test_merge_circRNA_detail.anno -l ../CIRI-vis_test/test_library_length.list -r test_ref.fa -min 1

...
...
>stout_82#chr1:3251039|3290753 length=420 12/205 +
>stout_82#chr1:3251039|3290753 length=519 10/205 +
>stout_83#chr1:3302871|3341317 length=479 53/72 +
>stout_83#chr1:3302871|3341317 length=637 18/72 +
>stout_85#chr1:3251039|3277912 length=306 4/5 +
>stout_86#chr1:2502478|2519276 length=523 21/21 +
chr1 Completed
Take time: 146s