Use RSEM and Bowtie2 to align paired-end sequences

Question

[melissachua90]

I want to use rsem-calculate-expression and bowtie2 aligner to align paired-end sequence based on the following conditions:

• 2 processors
• generate BAM file
• very fast bowtie2 sensitivity
• append gene/transcript name

My code:

rsem-refseq-extract-primary-assembly GCF_000001405.31_GRCh38.p5_genomic.fna GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna

rsem-prepare-reference --gff3 GCF_000001405.31_GRCh38.p5_genomic.gff --bowtie2 --bowtie2-path /bowtie2-2.4.5-py39hd2f7db1_2 --trusted-sources BestRefSeq GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna human_refseq


rsem-calculate-expression -p 2 --output-genome-bam --bowtie2 --bowtie2-sensitivity-level "very_fast" --append-names --paired-end s_1_1_sequence.txt s_1_2_sequence.txt human_refseq thyroid

Traceback:

[E::bgzf_flush] File write failed (wrong size)
Bus error
Bus error
(ERR): bowtie2-align exited with value 135
Bus error
"bowtie2 -q --phred33 --very-fast --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --no-discordant -p 2 -k 200 -x human_refseq -1 s_1_1_sequence.txt -2 s_1_2_sequence.txt | samtools view -S -b -o thyroid.temp/thyroid.bam -" failed! Plase check if you provide correct parameters/options for the pipeline!
Bus error

[ATpoint]

--bowtie2-path /bowtie2-2.4.5-py39hd2f7db1_2

You have to give the path to the bowtie2 binary, that here looks wrong. Or is this inside some kind of container?

[melissachua90]

I ran the rsem-calculate-expression from the home directory and all my files are also in the home directory.

[ATpoint]

Ok, but / is the root directory, not the current work directory. Maybe you mean ./ even though I suggest to use an absolute path instead.

[melissachua90]

You mean

rsem-prepare-reference --gff3 GCF_000001405.31_GRCh38.p5_genomic.gff --bowtie2 --bowtie2-path ./bowtie2-2.4.5-py39hd2f7db1_2 --trusted-sources BestRefSeq GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna human_refseq

?

Is there anything wrong with the subsequent code that might contribute to the error?

rsem-calculate-expression -p 2 --output-genome-bam --bowtie2 --bowtie2-sensitivity-level "very_fast" --append-names --paired-end s_1_1_sequence.txt s_1_2_sequence.txt human_refseq thyroid

[ATpoint]

1) yes, 2) i don't know, I am not a rsem user.