RNAseq pipeline (beginner level question): STAR alignment

[mlacencio]

Hello everyone!

After running the following command (via Snakemake):

STAR --genomeDir refs/STARindex --readFilesIn processing/transcriptomics/merged/S2_R1.fastq.gz processing/transcriptomics/merged/S2_R2.fastq.gz --readFilesCommand zcat --runThreadN 8 --outSAMtype BAM Unsorted --outFileNamePrefix processing/transcriptomics/alignment/S2/S2. --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000

I got the following files:

S2.Log.final.out
S2.Log.out
S2.Log.progress.out
S2.SJ.out.tab
S2.Aligned.out.bam
S2.sorted.bam.tmp.0108.bam
S2.sorted.bam.tmp.0107.bam
S2.sorted.bam.tmp.0106.bam
S2.sorted.bam.tmp.0105.bam
S2.sorted.bam.tmp.0104.bam
S2.sorted.bam.tmp.0103.bam
S2.sorted.bam.tmp.0102.bam
S2.sorted.bam.tmp.0101.bam
S2.sorted.bam.tmp.0100.bam
S2.sorted.bam.tmp.0099.bam
S2.sorted.bam.tmp.0098.bam
S2.sorted.bam.tmp.0065.bam
S2.sorted.bam.tmp.0032.bam

And the following warning:

[bam_sort] -m setting (500 bytes) is less than the minimum required (1M).

Trying to run with -m too small can lead to the creation of a very large number
of temporary files. This may make sort fail due to it exceeding limits on the
number of files it can have open at the same time.

Please check your -m parameter. It should be an integer followed by one of the
letters K (for kilobytes), M (megabytes) or G (gigabytes). You should ensure it
is at least the minimum above, and much higher if you are sorting a large file

Some questions:

Should I update my command according to the warning and perform the alignment process all over again? Or can I just continue from here as I have already the file "S2.Aligned.out.bam"? What does the presence of these temporary files "S2.sorted.bam.tmp.nnnn.bam" mean? How to proceed from here? This is the first time I run this pipeline!

Thank you very much in advance,

Marcio

[fchatonnet]

Dear Marcio,

it seems to me that the error you get comes from a samtools sort step done after the STAR alignment.
From your STAR command, you'll get an "unsorted" bam file (sorted by read name), and I guess your SnakeMake pipeline does apply a samtools sort step afterwards. It all depends what you want to do, but you can probably change the settings of the samtools sort step in the pipeline script. If you need a coordinate sorted bam file, you can generate if from the S2.Aligned.out.bam by applying samtools sort directly on it. Use the -@ and -m options to specify the numbers of threads (CPUs) and memory used, respectively. Or don't specify the -m parameter and it'll use 768 Mb of RAM by default.

Good luck!