no alignable.fq reads generated

[Maria Cartolano]

Hello,

I am trying to run rsem using STAR as aligner.

I am using the RSEM version 1.3.0 and following is my std.out

Basically it cannot find alignable.fq files since only unalienable.fq files are generated.

What could be the reason? 

/home/mcartola/Tools/STAR-2.5.2b/bin/Linux_x86_64_static/STAR --genomeDir /home/mcartola/RNAseqPipeline/HomoSapiensGRCh38  --outSAMunmapped Within  --outFilterType BySJout  --outSAMattributes NH HI AS NM MD  --outFilterMultimapNmax 20  --outFilterMismatchNmax 999  --outFilterMismatchNoverLmax 0.04  --alignIntronMin 20  --alignIntronMax 1000000  --alignMatesGapMax 1000000  --alignSJoverhangMin 8  --alignSJDBoverhangMin 1  --sjdbScore 1  --runThreadN 1  --genomeLoad NoSharedMemory  --outSAMtype BAM Unsorted  --quantMode TranscriptomeSAM  --outSAMheaderHD \@HD VN:1.4 SO:unsorted  --outFileNamePrefix Results/AS-136436.temp/AS-136436  --readFilesCommand zcat  --readFilesIn AS-136436-LR-20254_R1.fastq.gz AS-136436-LR-20254_R2.fastq.gz

Feb 01 05:17:11 ..... started STAR run

Feb 01 05:17:11 ..... loading genome

Feb 01 05:18:24 ..... started mapping

Feb 01 06:40:17 ..... finished successfully

rsem-parse-alignments /home/mcartola/RNAseqPipeline/HomoSapiensGRCh38/Hs10_star Results/AS-136436.temp/AS-136436 Results/AS-136436.stat/AS-136436 Results/AS-136436.temp/AS-136436.bam 3 -tag XM

Parsed 1000000 entries

Parsed 2000000 entries

Parsed 3000000 entries

Done!

rsem-build-read-index 32 1 0 Results/AS-136436.temp/AS-136436_alignable_1.fq Results/AS-136436.temp/AS-136436_alignable_2.fq

Cannot open Results/AS-136436.temp/AS-136436_alignable_1.fq! It may not exist.

"rsem-build-read-index 32 1 0 Results/AS-136436.temp/AS-136436_alignable_1.fq Results/AS-136436.temp/AS-136436_alignable_2.fq" failed! Plase check if you provide correct parameters/options for the pipeline!

Error in file(modelF, open = "r") : cannot open the connection

In addition: Warning message:

In file(modelF, open = "r") :

  cannot open file 'Results/AS-136436.stat/AS-136436.model': No such file or directory

Execution halted

I have enough space on my disk, so it cannot be due to the fact that not all temp files are not written.

Any help would be fully appreciated.

cheers

[Bo Li]

Hi Maria,

Could you check the first line of "Results/AS-136436.stat/AS-136436.cnt"
file? This line gives number of unalignable reads, number of alignable
reads and number of reads that are filtered out. If number of alignable
reads is 0, it means that STAR found no alignable reads.

Best,
Bo

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[Maria Cartolano]

Thank you for the swift reply.

Indeed, there are no mappable reads. From last night run, even the samples that "did work" have > 85% of unalienable reads.

I guess there is something wrong with the samples. 

cheers