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deweylab / RSEM

RSEM: accurate quantification of gene and isoform expression from RNA-Seq data
http://deweylab.biostat.wisc.edu/rsem/
GNU General Public License v3.0
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One of the mate is not paired-end! #158

Open MoLuLuMo opened 3 years ago

MoLuLuMo commented 3 years ago

Hi I used STARsolo for fastq (10X 3' V2) to generate bam file and then used resm for isoform quantification. But there is one issue: One of the mate is not paired-end!

rsem-parse-alignments GRCh38_ref/GRCh38_star/GRCh38 SC59_ISOFORM.temp/SC59_ISOFORM SC59_ISOFORM.stat/SC59_ISOFORM Aligned.toTranscriptome.out.bam 3 -tag XM Read NS500211:290:HH5TCBGX2:1:11106:19051:5252: One of the mate is not paired-end! (RSEM assumes the two mates of a paired-end read should be adjacent) "rsem-parse-alignments GRCh38_ref/GRCh38_star/GRCh38 SC59_ISOFORM.temp/SC59_ISOFORM SC59_ISOFORM.stat/SC59_ISOFORM Aligned.toTranscriptome.out.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

Here is my code

RSEM reference with fasta/gtf from cellranger

RSEM-1.3.3/rsem-prepare-reference \ --gtf refdata-cellranger-GRCh38-3.0.0/genes/genes.gtf \ --star --star-path STAR-2.7.7a/source\ -p 20 \ refdata-cellranger-GRCh38-3.0.0/fasta/genome.fa \ GRCh38_ref/GRCh38_star/GRCh38

run starsolo

STAR-2.7.7a/source/STAR --runThreadN 12 \ --genomeDir GRCh38_ref/GRCh38_star/ \ --soloType CB_UMI_Simple \ --soloCBwhitelist cellranger-3.1.0/cellranger-cs/3.1.0/lib/python/cellranger/barcodes/737K-august-2016.txt \ --soloUMIlen 10 \ --soloUMIfiltering MultiGeneUMI \ --soloCBmatchWLtype 1MM_multi_pseudocounts \ --quantMode TranscriptomeSAM\ --outSAMtype BAM SortedByCoordinate \ --readFilesIn SC59_S2_L001_R2_001.fastq,SC59_S2_L002_R2_001.fastq,SC59_S2_L003_R2_001.fastq,SC59_S2_L004_R2_001.fastq SC59_S2_L001_R1_001.fastq,SC59_S2_L002_R1_001.fastq,SC59_S2_L003_R1_001.fastq,SC59_S2_L004_R1_001.fastq

run RSEM

RSEM-1.3.3/rsem-calculate-expression -p 12 --paired-end \ --bam \ --estimate-rspd \ --append-names \ --output-genome-bam \ Aligned.toTranscriptome.out.bam \ GRCh38_ref/GRCh38_star/GRCh38 \ SC59_ISOFORM

pliu55 commented 3 years ago

I know samtools has a fixmate function. I never used it myself, but hopefully it could help to remove the unpaired mate. In addition, you may also try samtools view -F followed by a particular flag information to remove the unpaired mate.