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TreesLab / NCLscan

We have developed a new pipeline, NCLscan, which is rather advantageous in the identification of both intragenic and intergenic "non-co-linear" (NCL) transcripts (fusion, trans-splicing, and circular RNA) from paired-end RNA-seq data.
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Is free version of novoalign not adequate to NCLscan ? #9

Closed tyaoi closed 5 years ago

tyaoi commented 7 years ago

Hello, I just came about a trouble as the follows. Did the use of free version of novoalign cause this ? My PC environments is Ubuntu 14.04LTS. i7-6700K, 64GB RAM. All pre-required 5 tools were installed by using Linuxbrew that is equivalent to Homebrew in OSX. Moreover, NCLscan was run on Python 2.7.12 |Anaconda 4.2.0 (64-bit).

Sincerely,

Yaoi T

License file not found.

Licensed for evaluation, educational, and not-for-profit use only.

novoalign -r A 1 -g 99 -x 99 -d /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.JS.ndx -f /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.main.unmapped_1.fastq -o SAM

Starting at Thu Nov 3 20:17:47 2016

Interpreting input files as Sanger FASTQ.

Error: /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.JS.ndx does not appear to be a valid novoindex. Code 9

novoalign (V3.02.13 - Build Jun 29 2015 @ 12:27:18 - A short read aligner with qualities.

(C) 2008-2015 NovoCraft Technologies Sdn Bhd.

License file not found.

Licensed for evaluation, educational, and not-for-profit use only.

novoalign -r A 1 -g 99 -x 99 -d /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.JS.ndx -f /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.main.unmapped_2.fastq -o SAM

Starting at Thu Nov 3 20:17:47 2016

Interpreting input files as Sanger FASTQ.

Error: /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.JS.ndx does not appear to be a valid novoindex. Code 9 End of file reading 4 bytes Total time cost = 0.00139594078064 sec End of file reading 4 bytes Total time cost = 0.00144004821777 sec

tyaoi commented 7 years ago

Hello, I write another information in the below: In NCLscan.config, I wrote the PATH to tools bedtools_bin = /home/tyaoi4/.linuxbrew/bin/bedtools blat_bin = /home/tyaoi4/.linuxbrew/bin/blat bwa_bin = /home/tyaoi4/.linuxbrew/bin/bwa samtools_bin = /home/tyaoi4/.linuxbrew/bin/samtools novoalign_bin = /home/tyaoi4/.linuxbrew/bin/novoalign novoindex_bin = /home/tyaoi4/.linuxbrew/bin/novoindex

Sincerely,

Yaoi T

tyaoi commented 7 years ago

Hello,

I retried to run after obtaining trial license. However, the following error message was returned.

Error: /media/tyaoi4/SeaGate3TB/NCLscan_Results/CT11_NCLscan_Result/CT11_NCLscan.JS.ndx does not appear to be a valid novoindex. Code 9

What does " Code 9" mean ? How can I do to resolve this problem ?

I apprecite any advice. Thanks,

Yaoi T

chiangtw commented 7 years ago

Hi,

You may check the two files first,

Do these two files have normal file size?

Thanks!

tyaoi commented 7 years ago

Hi, Thanks for your quick response. Those files have 0 kb and 18.9 kb respectively. I don't know normal file size of them, but the size of "CT11_NCLscan.JS.fa" is probably not adequate.

Would you tell me the other check list ?

Thanks,

Yaoi T

chiangtw commented 7 years ago

Hi, Please check the file size of the following files,

Thanks!

tyaoi commented 7 years ago

Hi,

Thanks for your reply.

I attach the following files:

" output_name_size_list.txt " is the list of all produced files including each size.

" NCLscan.config " includes the actually used information. Herein, I conducted the runs by using mouse genome data files. Moreover, I tried a run by using the max_fragment_size = 1139 because the maximum in the fragment size distribution is shown to be from 1125 to 1139 (count 300). The max_read_len value is default because the read length is 90. However, I got the same results.

" Mus.genome.ref_name_size_list.txt " is the list of mouse reference genome that includes both the downloaded files and the files produced by the program.

Thanks,

矢追 毅 京都府立医科大学・院・分子病態病理学 助教 京都市上京区河原町通広小路梶井町465 TEL/FAX 075-251-5849

2016-11-14 8:07 GMT+09:00 Tai-Wei Chiang notifications@github.com:

Hi, Please check the file size of the following files,

  • CT11_NCLscan.sam
  • CT11_NCLscan.unmapped.sam
  • CT11_NCLscan.rG.psl
  • CT11_NCLscan.chi0.bed
  • CT11_NCLscan.chi.bed
  • CT11_NCLscan.preJS.bed
  • CT11_NCLscan.preJS.info2
  • CT11_NCLscan.JS.info
  • CT11_NCLscan.JS.cleaned.info
  • CT11_NCLscan.preJS.seq
  • CT11_NCLscan.preJS.seq2
  • CT11_NCLscan.preJS.seq12

Thanks!

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AllRef.fa 2950187399 AllRef.fa.amb 11343 AllRef.fa.ann 11449641 AllRef.fa.bwt 2894859392 AllRef.fa.pac 723714829 AllRef.fa.sa 1447429704 AllRef.ndx 8163709198 GRCm38.p4.genome.fa 2785778639 GRCm38.p4.genome.fa.fai 3255 RepChrM.fa 33151 gencode.vM11.annotation.gtf 810222289 gencode.vM11.lncRNA_transcripts.fa 20821697 gencode.vM11.pc_transcripts.fa 143553912

CT11_NCLscan.2.chi.bed 0 CT11_NCLscan.2.info 0 CT11_NCLscan.2.info.fa 0 CT11_NCLscan.2b.info 0 CT11_NCLscan.3.info 0 CT11_NCLscan.3b.info 0 CT11_NCLscan.4.info 0 CT11_NCLscan.4b.info 0 CT11_NCLscan.JS.GRCh37.2.psl 0 CT11_NCLscan.JS.cleaned.info 0 CT11_NCLscan.JS.clear.fa 0 CT11_NCLscan.JS.fa 0 CT11_NCLscan.JS.info 0 CT11_NCLscan.JS.info_1 0 CT11_NCLscan.JS.info_2 0 CT11_NCLscan.JS.ndx 16877 CT11_NCLscan.JS.seq 0 CT11_NCLscan.JS.seq12 0 CT11_NCLscan.JS.seq_1 0 CT11_NCLscan.JS.seq_2 0 CT11_NCLscan.JS1.Idx 0 CT11_NCLscan.JS1.preIdx 0 CT11_NCLscan.JS2.Idx 0 CT11_NCLscan.JS2.cleaned.info 0 CT11_NCLscan.JS2.fa 0 CT11_NCLscan.JS2.info 0 CT11_NCLscan.JS2.info_1 0 CT11_NCLscan.JS2.info_2 0 CT11_NCLscan.JS2.ndx 16877 CT11_NCLscan.JS2.preIdx 0 CT11_NCLscan.JS2.prefa 0 CT11_NCLscan.JS2.result.info 0 CT11_NCLscan.JS2.seq 0 CT11_NCLscan.JS2.seq_1 0 CT11_NCLscan.JS2.seq_2 0 CT11_NCLscan.PreJS2.bed 0 CT11_NCLscan.PreJS2.info 0 CT11_NCLscan.PreJS2.info2 2 CT11_NCLscan.PreJS2.seq 0 CT11_NCLscan.PreJS2.seq2 2 CT11_NCLscan.chi.bed 0 CT11_NCLscan.chi0.bed 0 CT11_NCLscan.chi2.bed 0 CT11_NCLscan.chi3.bed 0 CT11_NCLscan.colinear.psl 0 CT11_NCLscan.info 0 CT11_NCLscan.linearJS 0 CT11_NCLscan.main.JS2.sam 0 CT11_NCLscan.main.JS2b.sam 0 CT11_NCLscan.main.bwa.bam 106688204 CT11_NCLscan.main.bwa.unmapped.sam 12378112 CT11_NCLscan.main.bwa.unmapped.sam.id 0 CT11_NCLscan.main.bwa.unmapped_1.fastq 0 CT11_NCLscan.main.bwa.unmapped_2.fastq 0 CT11_NCLscan.main.sam 0 CT11_NCLscan.main.um3.fa 0 CT11_NCLscan.main.unmapped.fa 0 CT11_NCLscan.main.unmapped.sam 0 CT11_NCLscan.main.unmapped.sam.id 0 CT11_NCLscan.main.unmapped_1.fastq 0 CT11_NCLscan.main.unmapped_2.fastq 0 CT11_NCLscan.main_1.JS.sam 0 CT11_NCLscan.main_1.um3.fastq 0 CT11_NCLscan.main_2.JS.sam 0 CT11_NCLscan.main_2.um3.fastq 0 CT11_NCLscan.ncl.sam 0 CT11_NCLscan.preJS.bed 0 CT11_NCLscan.preJS.info 0 CT11_NCLscan.preJS.info2 2 CT11_NCLscan.preJS.seq 0 CT11_NCLscan.preJS.seq2 2 CT11_NCLscan.preResult 0 CT11_NCLscan.rG.non_un.psl 0 CT11_NCLscan.result.info 0 CT11_NCLscan.result.sam 0 CT11_NCLscan.result.tmp 0 CT11_NCLscan.tmp.info 0 CT11_NCLscan.tmp2.info 0 CT11_NCLscan.un.psl 0 CT11_NCLscan.unmapped.2.fa 0 CT11_NCLscan.unmapped.fa 0 all.CT11_NCLscan.1.result 0 all.CT11_NCLscan.1.sam 0 all.CT11_NCLscan.1b.result 0 all.CT11_NCLscan.1b.sam 0 all.CT11_NCLscan.JS.Parsered.sam 0 all.CT11_NCLscan.JS.sam 0 all.CT11_NCLscan.JS2.sam 0 all.CT11_NCLscan.JS2b.sam 0 all.CT11_NCLscan.b.result 0 all.CT11_NCLscan.result 0 all.CT11_NCLscan.um3.colinear.psl 0 all.CT11_NCLscan.um3.fa 0 temp.list 0 tmp.info 0

chiangtw commented 7 years ago

Hi,

It seems that there are no "unmapped reads" in the your input dataset. You may check these two files first:

"CT11_NCLscan.main.bwa.bam" was the alignment result mapped by bwa, and "CT11_NCLscan.main.bwa.unmapped.sam" was the unmapped part filtered by samtools.

tyaoi commented 7 years ago

Hi, Tai-Wei

Thank you for reply.

CT11_NCLscan.main.bwa.bam 106688204 CT11_NCLscan.main.bwa.unmapped.sam 12378112

I am sorry to trouble you, but thank you for your continuous support.

Yaoi T

chiangtw commented 7 years ago

I'm mean ... Are there some "unmapped-reads-removing" pre-processes on your input dataset? because it looks like that there are no unmapped reads in the dataset. NCLscan needs the unmapped reads to find the NCL candidates.

Thanks!