Problem converting bam to sam (samtools)

Question

[hinkel2]

Hi guys!

I'm new to RNAseq data analysis and related bioinformatic pipelines. I just aligned my PE-reads to genome using Tophat2:

tophat2 -p 4 -G ../Arabidopsis_thaliana.TAIR10.31.gtf ../Arabidopsis_thaliana.TAIR10.31.dna.genome PE.reads.1.fastq.gz PE.reads.2.fastq.gz

I then get the default output files: accepted_hits.bam etc.,

"alignment_summary.txt" says:

Left reads: Input: 37579106 Mapped : 35638921 (94.8% of input) of these: 703952 ( 2.0%) have multiple alignments (23 have >20) Right reads: Input : 37579106 Mapped : 32462213 (86.4% of input) of these: 614654 ( 1.9%) have multiple alignments (23 have >20) 90.6% overall read mapping rate.

Aligned pairs: 31803489 of these: 601459 ( 1.9%) have multiple alignments 5799 ( 0.0%) are discordant alignments 84.6% concordant pair alignment rate.

For some further downstream processing steps of my data I need sam instead of bam. That's why I wanted to use samtools to convert bam to sam (as already described in some threads here). I know that it is also possible to create a sam output by using "--no-convert-bam" but I forgot to add this in my tophat2 run.

So what I did: samtools view -h -o accepted_hits.sam accepted_hits.bam

Error message:

samtools view: writing to "accepted_hits_sam" failed: File too large samtools view: error closing "accepted_hits.sam": -1

I basically don't know why the file should be too large now. Did anyone face this problem before? Thanks in advance!

[GenoMax]

How about (if you are using an older version of samtools) : samtools view -h accepted_hits.bam > accepted_hits.sam

Answer 1

[Pierre Lindenbaum]

File too large

looks like a problem with your filesystem. e.g: http://stackoverflow.com/questions/730709/

[hinkel2]

Thank you!

I have a 64bit system (Ubuntu 16.04 LTS) on a dual boot machine. Is this file system problem due to the FAT32 and is there an easy way to fix it? Sorry for my noob questions.

[WouterDeCoster]

If I'm not mistaken FAT32 is limited to 4Gb files, which is likely too small for your sam file.

[Istvan Albert]

Two solutions come to mind:

1. Break you BAM file into two by filtering on a chromosome or by coordinates.
2. "Fake" the SAM file by streaming from the samtools view output.