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  • rsem-calculate-expression takes very long with paired-end reads (using bowtie2)

    I am want to use RSEM for transcript quantification. I have paired-end Illumina 1.5 encoded fastq files, a genome file, and a gtf file from Ensembl.
    First I used rsem-prepare-reference:


    rsem-prepare-reference --gtf myGTF.gtf \
    --bowtie2 \
    --bowtie2-path /my/path/bowtie2-2.2.3 \
    myGenome.fa \
    ./prepREF_bowtie2


    This works well (as far as I can tell) --- computation time: ~5min.
    Then I want to use rsem-calculate-expression:

    rsem-calculate-expression --phred64-quals \
    -p 10 \
    --time \
    --bowtie2 \
    --bowtie2-path /my/path/bowtie2-2.2.3 \
    --paired-end \
    ../RNA_reads/sample1_1.fq \
    ../RNA_reads/sample1_2.fq \
    ./prepREF_bowtie2 \
    ./Sample1_rsem


    I use a linux cluster with 10 CPUs and 10 GB memory. However all the output I get is the bowtie2 command line. I aborted the calculation after 30 hours. Why is this taking so long?
    (Using bowtie instead of bowtie2 seems to work: at least I am getting rsem-parse-alignments outputs. However bowtie was only able to align ~50% of the reads and I got some "Warning: Exhausted best-first chunk memory for read" warnings).
    What am I doing wrong?

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