Segmentation fault (core dumped) - during mapping with bacterial genome

[justin joseph jeyakani]

Hi Alex,

I'm mapping single end data to Enterobacter aerogenes genome, it contain only 1 reference sequence.

--genomeSAindexNbases  tried =LOG(5084929/2-1,2) 

--genomeChrBinNbits tried =LOG(5084929/1,2)

it doesn't work

The genome index created without any issue using the below command

--genomeSAindexNbases 5

--genomeChrBinNbits 6

STAR   --runMode genomeGenerate   --runThreadN 16   --genomeDir .   --genomeFastaFiles ../Enterobacteraerogenes.fa      --genomeSAindexNbases 5   --genomeChrBinNbits 6   --sjdbGTFfile ../gtf/Enterobacteraerogenes_annotation.gtf

ftp://ftp.ensemblgenomes.org/pub/bacteria/release-37/fasta/bacteria_90_collection/_enterobacter_aerogenes/dna/_enterobacter_aerogenes.ASM75554v1.dna_sm.toplevel.fa.gz

ftp://ftp.ensemblgenomes.org/pub/bacteria/release-37/gtf/bacteria_90_collection/_enterobacter_aerogenes/_enterobacter_aerogenes.ASM75554v1.37.gtf.gz

But when I run the star mapping after successfully load the genome it immediately exits with a segmentation fault. I tried with different no. the thread also.

 /mnt/projects/rpd/apps.testing/star-2.5.2a/bin/STAR --genomeDir /mnt/projects/rpd/genomes.testing/Enterobacter_aerogenes/star/ --runThreadN 4 --genomeLoad NoSharedMemory --readFilesCommand zcat --outFilterType BySJout --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --outSAMmapqUnique 50 --outSAMattributes NH HI AS nM NM MD  --outSAMstrandField intronMotif --outFilterMultimapNmax 1 --alignEndsTyp

e EndToEnd --outFileNamePrefix starout --readFilesIn /mnt/projects/userrig/solexa/HS003/HS003-SR-R00260_BCANT4ACXX/bcl2fastq_2017-09-11T20-07-04.244716/out/Project_MUX5656/Sample_RBB406/RBB406-GCCAAT_S30_L004_R1_001.fastq.gz

Sep 22 11:46:42 ..... started STAR run

Sep 22 11:46:42 ..... loading genome

Sep 22 11:46:42 ..... started mapping

Segmentation fault (core dumped)

Please find the attached log file.

Could you help me to fix this problem?

Thanks

Justin

[Alexander Dobin]

Hi Justin.

I generated genome with your parameters and mapped a few sequences extracted from the genome fasta, and it went without a problem.

Could you send me a small subset of reads that still causes the seg-fault?

Also, I would recommend not using --outFilterType BySJout  --outSAMstrandField intronMotif since the reads are not expected to be spliced.

Moreover, you may want to prohibit splicing altogether with --alignIntronMax 1

Cheers

Alex

[justin joseph jeyakani]

Hi Alex,

Thank you for helping me.

I do try with the parameters you mentioned it didn't work, it works if I don't use the "TranscriptomeSAM" (--quantMode TranscriptomeSAM GeneCounts). Please let me know how to fix, hence I need to run rsem with the transcriptome bam.

I have attached small subset of the fastq file.

Please let me know the parameter you used to create the index for this genome?

I created with --genomeChrBinNbits and --genomeSAindexNbases 6. The below formula I tried in the excel gives 22 and 

--genomeChrBinNbits

=LOG(5084929/1,2)

--genomeSAindexNbases

=LOG(5084929/2-1,2)

The following command runs without any issue.

/mnt/projects/rpd/apps.testing/star-2.5.2a/bin/STAR --genomeDir /mnt/projects/rpd/genomes.testing/Enterobacter_aerogenes/star/ --outSAMattrRGline ID:1c481aa9 PL:Illumina LB:RBB406 SM:RBB406 CN:GIS --runThreadN 1 --genomeLoad NoSharedMemory --readFilesCommand zcat --outFilterType BySJout --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --outSAMmapqUnique 50 --outSAMattributes NH HI AS nM NM MD  --outSAMstrandField intronMotif --outFilterMultimapNmax 1 --alignEndsType EndToEnd --alignIntronMax 1 --outFileNamePrefix starout --readFilesIn /mnt/projects/userrig/solexa/HS003/HS003-SR-R00260_BCANT4ACXX/bcl2fastq_2017-09-11T20-07-04.244716/out/Project_MUX

5656/Sample_RBB406/RBB406-GCCAAT_S30_L004_R1_001.fastq.gz

Thanks

Justin

[Alexander Dobin]

Hi Justin,

I have used:

$ STAR --runThreadN 12 --genomeSAindexNbases 5 --genomeChrBinNbits 6 --runMode genomeGenerate --genomeDir ./ --genomeFastaFiles _enterobacter_aerogenes.ASM75554v1.dna_sm.toplevel.fa --sjdbGTFfile _enterobacter_aerogenes.ASM75554v1.37.gtf

and

$ STAR --genomeDir ./GenomeDir --runThreadN 4 --genomeLoad NoSharedMemory --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --outSAMmapqUnique 50 --outSAMattributes NH HI AS nM NM MD --outSAMstrandField intronMotif --outFilterMultimapNmax 1 --alignEndsType EndToEnd --readFilesIn RBB406-GCCAAT_S30_L004_R1_001.fastq

and they completed without problems. I am using 2.5.3a, please try it as well just it case.

If running these commands still causes the problem, please send me the Log.out files from both the genome generation and mapping steps.

Cheers

Alex

On Friday, September 22, 2017 at 4:10:06 AM UTC-4, justin joseph jeyakani wrote: