NCLscan finishes with an empty result file

[HosseinAsghari]

Hi,

I am trying NCLscan on some paired-end data and the run finishes with the following message:

The result will be written to out.result
See out.result.sam for the final alignment result.

However, "out.result" is an empty file while I expect to get many circRNAs.

At the end of the standard error output I got a bunch of "Format Error!" and a few "Format Error! Not paired-end!".
Here is the list of output files:
output.list.txt

I was wondering what would be the reason of this issue.

Thanks in advance,
Hossein

[chiangtw]

Hi,

Please provide the following information:

• the version of NCLscan
• the version of Novoalign
• the complete error message (if possible)
• the read length of the input dataset
  • Is it paired-end? or single-end?

thanks,
tw

[HosseinAsghari]

Hi,

• NCLscan V1.6.5 (latest release)
• Novoalign V4.00.01
• 2x101 paired-end

Log files:
ncls.log.txt
ncls.out.txt

Hossein

[chiangtw]

Hi,

If you don't mind, please send me the file "all.out.JS2.sam" in the NCLscan output directory.

And could you try the test dataset mentioned in the README?
I was wondering if it could be run successfully.

the following is the link to the test dataset:

• ftp://treeslab1.genomics.sinica.edu.tw/NCLscan/test.tar.gz

Thanks,
tw

[HosseinAsghari]

Hi again and thanks for the follow up.

Here is the file you requested.

all.out.JS2.sam.zip

And yes, I have tested the sample dataset. It runs without any problems and produces results.

Hossein

[chiangtw]

Hi,

This issue should be due to the behavior changes of novoalign V4.

The option "--pechimera", which is used to enable the use of supplementary alignments when one read of a pair is chimeric, changed its default value to "on".

This change would break the NCLscan pipeline.

The following was found in the file "all.out.JS2.sam",

simulate:8826	99	simulate:8847.0	89	0	63S38M	=	275	287	TTCGGCCTCTACCCAAAGTGAAAAGACTGCTGTCAGATAGCACTTGCCTTCCCCATATTATTCAGAAAAATGTTAAGGATCTCATGGATTCAAATGGAATA	55555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555	PG:Z:novoalign	AS:i:0	UQ:i:0	NM:i:0	MD:Z:38	PQ:i:8	SM:i:0	AM:i:0	ZS:Z:R	NH:i:10	HI:i:1	IH:i:1	Z3:i:126	SA:Z:simulate:8847.6,931,+,63M38S,3,0;
simulate:8826	147	simulate:8847.0	275	0	101M	=	89	-287	GTGGCCCGTATGGATTTCATGAGATGCAAGAATTGTGGACCAAAGGAATGTTAAATGCAAAAACCAGATGCTGGGCTCAAGGCATGGATGGATGGCGACCA	55555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555	PG:Z:novoalign	AS:i:0	UQ:i:0	NM:i:0	MD:Z:101	PQ:i:8	SM:i:0	AM:i:0	ZS:Z:R	NH:i:10	HI:i:1	IH:i:1	Z3:i:375
simulate:8826	2145	simulate:8847.6	931	3	63M38S	simulate:8847.0	275	0	TTCGGCCTCTACCCAAAGTGAAAAGACTGCTGTCAGATAGCACTTGCCTTCCCCATATTATTCAGAAAAATGTTAAGGATCTCATGGATTCAAATGGAATA	55555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555555	PG:Z:novoalign	AS:i:0	UQ:i:0	NM:i:0	MD:Z:63	ZS:Z:R	NH:i:10	HI:i:1	IH:i:1	Z3:i:993	SA:Z:simulate:8847.0,89,+,63S38M,0,0;

The parser in NCLscan pipeline needs these alignments to be ordered by pairs,
but these additional rows would break it.

--

For now, you could just use novoalign V3 to avoid this problem.

And we should fix this in the future.

Thanks!
tw