Hi guys

I am now running RSEM to do a transcript quantification and become stuck on the quantification step.

Here is the command line I used

rsem-calculate-expression --paired-end --star --star-gzipped-read-file --sort-bam-by-read-name --output-genome-bam --paired-end -p 8 1A_1.fastq.gz 1A_2.fastq.gz reference 1A

The terminal then returned

STAR --genomeDir .  --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 8  --genomeLoad NoSharedMemory  --outSAMtype BAM Unsorted  --quantMode TranscriptomeSAM  --outSAMheaderHD @HD VN:1.4 SO:unsorted  --outFileNamePrefix 1A.temp/1A  --readFilesCommand zcat  --readFilesIn 1A_1.fastq.gz 1A_2.fastq.gz
    STAR --genomeDir . --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 8 --genomeLoad NoSharedMemory --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --outSAMheaderHD @HD VN:1.4 SO:unsorted --outFileNamePrefix 1A.temp/1A --readFilesCommand zcat --readFilesIn 1A_1.fastq.gz 1A_2.fastq.gz
    STAR version: 2.7.9a   compiled: 2021-05-04T09:43:56-0400 vega:/home/dobin/data/STAR/STARcode/STAR.master/source
Jun 09 10:27:35 ..... started STAR run
Jun 09 10:27:35 ..... loading genome
Jun 09 10:28:05 ..... started mapping
Jun 09 12:10:19 ..... finished mapping
Jun 09 12:10:20 ..... finished successfully

samtools sort -n -@ 8 -m 1G -o 1A.temp/1A.sorted.bam 1A.temp/1A.bam

rm -f 1A.temp/1A.bam

rsem-parse-alignments reference 1A.temp/1A 1A.stat/1A 1A.temp/1A.sorted.bam 3 -tag XM
"rsem-parse-alignments reference 1A.temp/1A 1A.stat/1A 1A.temp/1A.sorted.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!
Thu Jun  9 12:36:06 HKT 2022
Thu Jun  9 12:36:11 HKT 2022

So does that means the input files "1A_1.fastq.gz" and "1A_2 fastq.gz" are incompatible with the "reference" files generated by the rsem-prepare-reference command line?

I also received a warning when running rsem-calculate-expression:

RSEM/1.3.3 is loaded 
STAR/2.7.9a is loaded 
[bam_sort_core] merging from 288 files and 8 in-memory blocks...
Warning: The SAM/BAM file declares less reference sequences (178790) than RSEM knows (179372)! Please make sure that you aligned your reads against transcript sequences instead of genome.
Read A00549:165:HY77CDSXY:2:1101:1045:2895: The adjacent two lines do not represent the two mates of a paired-end read! (RSEM assumes the two mates of a paired-end read should be adjacent)

Does this mean I have to do some modifications on my gtf file? To omit some of the extra reference sequences?

Thanks