Bowtie2 parameter error.

[Christian Hodar]

Hi,

I have problems with bowtie2 step in miRNA pipeline, with the index generation. The error is the next:

[Fri Jun 29 11:19:17 2018] Indexing /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta for a bowtie2 analysis [Fri Jun 29 11:19:17 2018] Generating the index genome dmel.flybase from /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta. This process could take some hours BOWTIE2_INDEX ERROR :: system args failed: 256 (bowtie2-build --threads 20 -f /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//Bowtie2_index/dmel.flybase) at lib/CbBio/RNASeq/Aligner.pm line 1327.

I check the log file and show this: Error: Encountered internal Bowtie 2 exception (#1) Command: bowtie2-build --wrapper basic-0 --threads 20 -f /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//Bowtie2_index/dmel.flybase

The index options in my ini file are: [Aligner] aligner=bowtie2 dir=/mnt/Raid_Dell/Dmel.genome/fastas.flybase/ fasta=/mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta logfile=bowtie.dmel.log indexname=dmel.flybase threads=20

Any help is welcome, thanks. Christian.

[Eduardo Andres Leon]

Dear Christian, As I see you did not use the specific parameters to create an index file (fasta and indexname). Please use this parameters as an example:

[Aligner]
; Aligner (Bowtie1, Bowtie2, BWA, miRDeep or Bowtie1-Bowtie2)
aligner=Bowtie2
; Path of the genomic fasta sequence to build the index
fasta=Genomes/Indexes/bowtie2/human/homo_sapiens19.fa
;Name to write in the index files
indexname=bw2_homo_sapiens19

E

[Christian Hodar]

Hi Eduardo, But the parameters are there:

[Aligner] 
aligner=bowtie2
dir=/mnt/Raid_Dell/Dmel.genome/fastas.flybase/ 
fasta=/mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta
indexname=dmel.flybase 
threads=20

[Eduardo Andres Leon]

Yes, but you are mixing parameters from the section [General] with parameters from the [Aligner] section.

Threads and dir are from [General], so they are ignored. Use this an example: https://bitbucket.org/cbbio/miarma/src/master/Examples/basic_examples/miRNAs/Known_miRNAs/3.Aligner/3.2.Bowtie2_index_from_fasta.ini

[Christian Hodar]

Hi Eduardo,

I move the ini file to this, removing the duplicate parameters already described in General:

[General]
type=miRNA
read_dir=/mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/
label=Dietas_map
miARmaPath=.
output_dir=/mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results/
organism=dmelanogaster
threads=20
strand=yes
logfile=/mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results/run_log.log
stats_file=/mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results/miARma_stat.871.log

[Quality]
prefix=Both

[Adapter]
adapter=AGATCGGAAGAG
adaptersoft=CutAdapt

[Aligner]
aligner=Bowtie2
fasta=/mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta
indexname=dmel.flybase

[ReadCount]
database=/mnt/Raid_Dell/Dmel.genome/gff.flybase/test.gtf
seqid=gene_id
featuretype=exon

But still have the error:

[Sun Jul  1 18:14:15 2018] Generating the index genome dmel.flybase from /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta. This process could take some hours
BOWTIE2_INDEX ERROR :: system args failed: 256 (bowtie2-build --threads 20 -f /mnt/Raid_Dell/Dmel.genome/fastas.flybase/dmel-all-chromosome-r6.21.fasta /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//Bowtie2_index/dmel.flybase) at lib/CbBio/RNASeq/Aligner.pm line 1327.

[Eduardo Andres Leon]

Would you mind to include both logs (logfile and stats_file ?

Thanks in advance

[Christian Hodar]

Hi Eduardo. Here attach the two requested files. Thanks, Christian.

[Eduardo Andres Leon]

Hi, I've seen that your error has been marked as a bug by bowtie2 (https://github.com/BenLangmead/bowtie2/issues/36). For whatever reason the thread option is not working properly, some users said that odd numbers din't work, other even number did not work. could you try using just one thread ?

E

[Christian Hodar]

Hi Eduardo, I try with one thread and pass the bowtie step. Another error was found in featurecounts, probably in some missing parameter (i didn't check yet). Here the output.

SEQCOUNT ERROR :: system args failed: 65280 (mkdir -p /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//cut_bw2_readcount_results/ ;featureCounts  -s 1 -t exon -g gene_id -T 1 -a /mnt/Raid_Dell/Dmel.genome/gff.flybase/test.gtf -o /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//cut_bw2_readcount_results/HS-2_S2_L001_R1_001_cut_bw2.tab /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results//Bowtie2_results/HS-2_S2_L001_R1_001_cut_bw2.sam 2>> /mnt/Raid_Dell/RNAseq.Dietas.Dmel/smRNA/Known_miRNAs/results/run_log.log) at lib/CbBio/RNASeq/Readcount.pm line 200, <STAT> line 318.

Christian.

[Christian Hodar]

I also try to change the binaries file from bowtie2 in bin/bowtie2 folder with a version without bug, but maybe the required parameters are not the same:

[Wed Jul  4 14:27:58 2018] Checking Aligner-bowtie2index parameter ... error!
    ERROR Please check that the parameter bowtie2index inside section [Aligner] is correct.

[Eduardo Andres Leon]

For both errors I'd need to check the log files and the ini file. But there is a strange thing in the second one: once miARma creates the index (1 thread), it writes the bowtie2index value within the ini file and starts the alignenmet step (by the way as you have the index created you can use now 20 threads to do the alignment). So the error that I see is related to this bowtieindex path (no related with the bowtie binary). Did you modify/move the files inside the Bowtie2_results folder ?

[Eduardo Andres Leon]

Any update ?

[Christian Hodar]

Uh. Sorry. I will check today in a couple of hours and let you know. Best regards, Christian

[Christian Hodar]

I check the log file and I guess that the problem is this:

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file /mnt/Raid_Dell/Dmel.genome/gff.flybase/test.gtf ...   ||
||    Features : 0                                                            ||
|| WARNING no features were loaded in format GTF.                             ||

So I will check my GTF before try again. I will you comment later. Thanks.

[Eduardo Andres Leon]

How was this new test ?

[Christian Hodar]

Hi Eduardo,

Sorry, I had to make an unespected trip and I I will not return to the office until Tuesday. Once I'm there I try again. All the best,