error on rsem running with mouse

[JiheyKim]

I got an error when running rsem with mouse.

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/cm/shared/apps/STAR/2.7.10a/STAR --genomeDir /mnt/beegfs/kimj23/rsem_genome/rsem_ref_mm10 --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 16 --genomeLoad NoSharedMemory --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --outSAMheaderHD @hd VN:1.4 SO:unsorted --outFileNamePrefix ./rsem_out/test.temp/test --readFilesCommand zcat --readFilesIn SRR6067904_1.fastq.gz SRR6067904_2.fastq.gz
/cm/shared/apps/STAR/2.7.10a/STAR --genomeDir /mnt/beegfs/kimj23/rsem_genome/rsem_ref_mm10 --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 16 --genomeLoad NoSharedMemory --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --outSAMheaderHD @hd VN:1.4 SO:unsorted --outFileNamePrefix ./rsem_out/test.temp/test --readFilesCommand zcat --readFilesIn SRR6067904_1.fastq.gz SRR6067904_2.fastq.gz
STAR version: 2.7.10a compiled: 2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source
Oct 04 09:45:52 ..... started STAR run
Oct 04 09:45:52 ..... loading genome
Oct 04 09:46:09 ..... started mapping
Oct 04 09:47:01 ..... finished mapping
Oct 04 09:47:03 ..... finished successfully

rsem-parse-alignments /mnt/beegfs/kimj23/rsem_genome/rsem_ref_mm10/rsem_ref_mm10 ./rsem_out/test.temp/test ./rsem_out/test.stat/test ./rsem_out/test.temp/test.bam 3 -tag XM
Parsed 1000000 entries
Done!

rsem-build-read-index 32 1 0 ./rsem_out/test.temp/test_alignable_1.fq ./rsem_out/test.temp/test_alignable_2.fq
Build Index ./rsem_out/test.temp/test_alignable_1.fq is Done!
Build Index ./rsem_out/test.temp/test_alignable_2.fq is Done!

rsem-run-em /mnt/beegfs/kimj23/rsem_genome/rsem_ref_mm10/rsem_ref_mm10 3 ./rsem_out/test ./rsem_out/test.temp/test ./rsem_out/test.stat/test -p 16 -b ./rsem_out/test.temp/test.bam 0 --append-names
Refs.loadRefs finished!
Thread 0 : N = 30, NHit = 80
Thread 1 : N = 26, NHit = 79
Thread 2 : N = 31, NHit = 81
Thread 3 : N = 33, NHit = 78
Thread 4 : N = 29, NHit = 81
Thread 5 : N = 34, NHit = 79
Thread 6 : N = 35, NHit = 80
Thread 7 : N = 36, NHit = 79
Thread 8 : N = 35, NHit = 80
Thread 9 : N = 30, NHit = 78
Thread 10 : N = 35, NHit = 80
Thread 11 : N = 33, NHit = 79
Thread 12 : N = 34, NHit = 78
Thread 13 : N = 29, NHit = 80
Thread 14 : N = 31, NHit = 78
Thread 15 : N = 20, NHit = 58
EM_init finished!
1000000 READS PROCESSED
estimateFromReads, N0 finished.
estimateFromReads, N1 finished.
The alignment of fragment SRR6067904.2933779 to transcript 35 starts at -80 from the forward direction, which should be a non-negative number! It is possible that the aligner you use gave different read lengths for a same read in SAM file.
"rsem-run-em /mnt/beegfs/kimj23/rsem_genome/rsem_ref_mm10/rsem_ref_mm10 3 ./rsem_out/test ./rsem_out/test.temp/test ./rsem_out/test.stat/test -p 16 -b ./rsem_out/test.temp/test.bam 0 --append-names" failed! Plase check if you provide correct parameters/options for the pipeline!

[hmgene]

nested exon problem considered here
alexdobin/STAR#1128

[hmgene]

proposed solution

1. try sort -u <old.gtf> > <new.gtf> and then regenerate index
2. simple script to ignore transcripts having overlapping exons.

[JiheyKim]

Thanks so much!
sort -u works!
Super thanks!!

[hmgene]

나도 깜놀 던져본건데 사랑해

…

Sent from my iPhone

On Oct 4, 2022, at 11:45, Jihye Kim ***@***.***> wrote:  Thanks so much! sort -u works! Super thanks!! — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you commented.

[JiheyKim]

그냥 던저봤다니.. 내가 깜놀.
나도 사랑해 히히히~