STAR-RNA Segmentation Fault

Question

[Poetic_Premium6]

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

[Bastien Hervé]

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 ?

Answer 1

[Bastien Hervé]

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

[Poetic_Premium6]

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!

[Bastien Hervé]

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

[Poetic_Premium6]

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

[Bastien Hervé]

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

[Poetic_Premium6]

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?

[Bastien Hervé]

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

[Poetic_Premium6]

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

[Bastien Hervé]

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

[Poetic_Premium6]

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!!!

[Bastien Hervé]

Maybe your fastq files are corrupt.

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

[Poetic_Premium6]

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

Answer 2

[Jeffin Rockey]

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.

[Poetic_Premium6]

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