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STAR-RNA Segmentation Fault

Hey all, I've been stalking this website for a few months for help (with success), and now I've run into my first problem that I can't solve :O . I am a wet-biologist by training so I'm still a noob at bioinformatics!

I am using STAR for E. coli reads and lately I have been receiving "segmentation Fault" error during the mapping portion. I have about 25 Gb of free RAM and read/write/execute permissions all set for user. I am using Debian and tried using both STAR versions2.5 & 2.6 I do not think anything is wrong with my code... STAR --runThreadN 3 --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --sjdbGTFfile /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_annotation.gtf --sjdbOverhang 100 --readFilesIn /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim1.fastq /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim2.fastq --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate Unsorted

Does anyone have a fix or some suggestions so I may continue troubleshooting?

Thanks for your time @Biostars, ~Jonathan J.S

rna-seq star mapping debian segmentation error
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updated by Jeffin Rockey Jeffin Rockey • written by Poetic_Premium6 • Poetic_Premium6

Could you copy/paste the log file involved please.

Do not use --readFilesCommand zcat if your files are not compressed

Did your index creation end succesfully ?

What was your command to create your index file ?

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written by Bastien Hervé Bastien Hervé

2 answers

Seems like you mixed up the two commands.

First create the index

STAR --runThreadN 3 --runMode genomeGenerate --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --genomeFastaFiles /path/to/reference/genome/Escherchia_coli_UTI89_reference_genome.fasta --sjdbGTFfile /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_annotation.gtf --sjdbOverhang 100

Then, process to the alignment

STAR --runThreadN 3 --runMode alignReads --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --readFilesIn /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim1.fastq /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim2.fastq --outSAMtype BAM SortedByCoordinate Unsorted
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written by Bastien Hervé Bastien Hervé

Hey Bastien, thank you for your clarification in splitting the commands. This is way is cleaner for sure! While the genome generation has not been a problem, I am still receiving the "segmentation fault" error the alignment portion.

Additionally, I think that the --readFilesCommand zcat must follow the rna-seq FASTQ files? Regardless, do you have any further suggestions? Thanks for your time!

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written by Poetic_Premium6 • Poetic_Premium6

Could you please execute the following command and copy/paste the result below :

ls -alt /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/

--readFilesCommand UncompressionCommand option, where UncompressionCommand is the un-compression command that takes the file name as input parameter, and sends the uncompressed output to stdout. For example, for gzipped files (*.gz) use --readFilesCommand zcat OR --readFilesCommand gunzip -c. For bzip2-compressed files, use --readFilesCommand bunzip2 -c

As you have UTI89_PostTrim1.fastq and not UTI89_PostTrim1.fastq.gz, you do not need --readFilesCommand

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written by Bastien Hervé Bastien Hervé

Good point, as it is redundant! Sure, here are the results:

-rw-rw-rw- 1 jonathan7 jonathan7 19483 Jun 4 09:53 Log.out

-rw-rw-rw- 1 jonathan7 jonathan7 236 Jun 4 09:53 Log.progress.out

drwxrwxrwx 10 jonathan7 jonathan7 4096 Jun 4 09:53 .

-rw-rw-rw- 1 jonathan7 jonathan7 0 Jun 4 09:53 Aligned.out.bam

-rw-rw-rw- 1 jonathan7 jonathan7 0 Jun 4 09:53 Aligned.sortedByCoord.out.bam

drwx------ 3 root root 4096 Jun 4 09:53 _STARtmp

-rw-rw-rw- 1 jonathan7 jonathan7 1565873619 Jun 4 09:46 SAindex

-rw-rw-rw- 1 jonathan7 jonathan7 42734764 Jun 4 09:46 SA

-rw-rw-rw- 1 jonathan7 jonathan7 2774 Jun 4 09:46 exonGeTrInfo.tab

-rw-rw-rw- 1 jonathan7 jonathan7 864 Jun 4 09:46 exonInfo.tab

-rw-rw-rw- 1 jonathan7 jonathan7 1031 Jun 4 09:46 geneInfo.tab

-rw-rw-rw- 1 jonathan7 jonathan7 5505024 Jun 4 09:46 Genome

-rw-rw-rw- 1 jonathan7 jonathan7 870 Jun 4 09:46 genomeParameters.txt

-rw-rw-rw- 1 jonathan7 jonathan7 6 Jun 4 09:46 sjdbInfo.txt

-rw-rw-rw- 1 jonathan7 jonathan7 0 Jun 4 09:46 sjdbList.fromGTF.out.tab

-rw-rw-rw- 1 jonathan7 jonathan7 0 Jun 4 09:46 sjdbList.out.tab

-rw-rw-rw- 1 jonathan7 jonathan7 4268 Jun 4 09:46 transcriptInfo.tab

-rw-rw-rw- 1 jonathan7 jonathan7 15 Jun 4 09:46 chrLength.txt

-rw-rw-rw- 1 jonathan7 jonathan7 39 Jun 4 09:46 chrNameLength.txt

-rw-rw-rw- 1 jonathan7 jonathan7 24 Jun 4 09:46 chrName.txt

-rw-rw-rw- 1 jonathan7 jonathan7 18 Jun 4 09:46 chrStart.txt

drwx------ 2 root root 4096 Jun 4 09:37 _STARgenome

drwxrwxrwx 5 jonathan7 jonathan7 4096 Jun 4 04:52 ..

-rw-rw-r-- 1 jonathan7 jonathan7 134 Jun 1 04:08 genes.fpkm_tracking

-rw-rw-r-- 1 jonathan7 jonathan7 134 Jun 1 04:08 isoforms.fpkm_tracking

-rw-rw-r-- 1 jonathan7 jonathan7 0 Jun 1 04:08 skipped.gtf

-rw-rw-r-- 1 jonathan7 jonathan7 0 Jun 1 04:08 transcripts.gtf

-rw-rw-rw- 1 jonathan7 jonathan7 206422016 May 31 09:34 UTI89_PostTrim2.fastq.gz

-rw-rw-rw- 1 jonathan7 jonathan7 0 May 24 04:59 G15489_htseq_counts_pos_sorted.out

-rw-rw-rw- 1 jonathan7 jonathan7 32 May 24 04:59 accepted_hits_uniq_sorted.bam.bai

-rw-rw-rw- 1 jonathan7 jonathan7 358 May 24 04:59 accepted_hits_uniq_sorted.bam

-rw-rw-rw- 1 jonathan7 jonathan7 348 May 24 04:59 accepted_hits_uniq.bam

-rw-rw-rw- 1 jonathan7 jonathan7 32 May 24 04:59 Aligned.bam.bai -rw-rw-rw- 1 jonathan7 jonathan7 1803 May 23 07:47 Log.final.out

-rw-rw-rw- 1 jonathan7 jonathan7 0 May 23 07:47 SJ.out.tab

-rw-rw-rw- 1 jonathan7 jonathan7 348 May 23 07:31 Aligned.bam

-rw-rw-rw- 1 jonathan7 jonathan7 777 May 22 10:10 Aligned.out.sam

-rw-rw-rw- 1 jonathan7 jonathan7 626458 May 18 05:14 UTI89_annotation.gtf

drwxrwxrwx 2 jonathan7 jonathan7 4096 May 18 04:54 genomeDir

-rw-rw-rw- 1 jonathan7 jonathan7 5244844 May 18 04:30 UTI_genomic.fasta

-rw-rw-rw- 1 jonathan7 jonathan7 311377111 Apr 30 05:19 UTI89_PostTrim1.fastq.gz

-rw-rw-rw- 1 jonathan7 jonathan7 1002466424 Apr 30 05:19 UTI89_PostTrim1.fastq 

-rw-rw-rw- 1 jonathan7 jonathan7 1285209680 Apr 30 04:57 UTI89_PostTrim2.fastq 

-rw-rw-rw- 1 jonathan7 jonathan7 3893 Apr 24 08:35 index.html

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written by Poetic_Premium6 • Poetic_Premium6

Thanks, and in the Log files what do you have ?

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written by Bastien Hervé Bastien Hervé

Oh lovely, it is just a fatal input error now based on a read input rather than a "Segmentation Fault". Anyway, the log.out file exceeds the character limitation, which specific portions are necessary for diagnostics?

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written by Poetic_Premium6 • Poetic_Premium6

Oh lovely, it is just a fatal input error now based on a read input rather than a "Segmentation Fault"

You are tilting me OP :), you have fastq.gz not fastq, look :

-rw-rw-rw- 1 jonathan7 jonathan7 311377111 Apr 30 05:19 UTI89_PostTrim1.fastq.gz

-rw-rw-rw- 1 jonathan7 jonathan7 206422016 May 31 09:34 UTI89_PostTrim2.fastq.gz

Use this :

STAR --runThreadN 3 --runMode alignReads --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --readFilesIn /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim1.fastq.gz /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim2.fastq.gz --readFilesCommand gunzip -c --outSAMtype BAM SortedByCoordinate Unsorted

--readFilesCommand gunzip -c or --readFilesCommand zcat as you wish, both seems to work

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written by Bastien Hervé Bastien Hervé

Haha sorry for the tilt, as I mentioned in the header... I am a noob :P .

Besides, I kept both files w/ & w/o gzip in the directory just to try to see if it would result in a memory difference and must have confused them in the original post but..... I tried both options from your new command with the gz files and they still lead back to Segmentation Fault again. 

STAR --runThreadN 3 --runMode alignReads --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --readFilesIn /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim1.fastq.gz /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim2.fastq.gz --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate Unsorted
• 1 views • link
written by Poetic_Premium6 • Poetic_Premium6

I got this error once, problem with gzipped file. So last try.

Remove this parameter --readFilesCommand zcat

And decompress your UTI89_PostTrim1.fastq.gz and UTI89_PostTrim2.fastq.gz

Then retry, something like this :

STAR --runThreadN 3 --runMode alignReads --genomeDir /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/ --readFilesIn /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim1.fastq /home/jonathan7/Documents/Comp_Micro/STAR/Escherchia_coli_UTI89/UTI89_PostTrim2.fastq --outSAMtype BAM SortedByCoordinate Unsorted
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written by Bastien Hervé Bastien Hervé

Bastien, I tried this change too without any luck. I think that I will have to reach out to the software developer, Alex for help.

Thank again for all of your help and time, it was greatly appreciated!!!

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written by Poetic_Premium6 • Poetic_Premium6

Maybe your fastq files are corrupt.

Good luck for your investigation and don't forget to put the answer below if there is one

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written by Bastien Hervé Bastien Hervé

Thanks Bastien, I assumed that too and re-did QC to check. I'll post if anything comes up. Best wishes!

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written by Poetic_Premium6 • Poetic_Premium6

Most likely the genome being small would be the problem. I had encountered seg-fault issue while dealing with very small genomes. Pasted below is a snippet from section 2.2.5 of STAR 2.4 manual. Please see whether setting the parameter as advised would solve the issue.

Very small genome.

For small genomes, the parameter --genomeSAindexNbases needs to be scaled down, with a typical value of min(14, log2(GenomeLength)/2 - 1). For example, for 1 megaBase genome, this is equal to 9, for 100 kiloBase genome, this is equal to 7.

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written by Jeffin Rockey Jeffin Rockey

Hey Jeffin, I just tried this without any success in fixing the segmentation fault. But surely, you are correct...this would be great to keep in my code :P

Best, ~Jonathan J.S

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written by Poetic_Premium6 • Poetic_Premium6

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