Question: rsem-calculate-expression error message
0
gravatar for shuksi1984
18 months ago by
shuksi198450
shuksi198450 wrote:

My RNAseq pipeline has following steps:

STEP1-extract primary assembly:

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

STEP2-preapare reference:

path/to/rsem-prepare-reference --gff3
 GCF_000001405.31_GRCh38.p5_genomic.gff --trusted-sources
 BestRefSeq,Curated\ Genomic --bowtie2
 GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna
 human.refseq.gff

STEP3-Indexing with star:

path/to/STAR --runThreadN 2 --runMode
genomeGenerate --genomeDir /path/to/genome_file --genomeFastaFiles
path/to/GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna
--sjdbGTFfile path/to/human.refseq.gff --sjdbOverhang 100 --limitGenomeGenerateRAM=8825541333 --genomeSAsparseD 2

STEP4-calculate expression using STAR:

path/to/rsem-calculate-expression -p 2 --output-genome-bam --star
--star-path path/to/STAR --paired-end path/to/SRR925687_1.fastq path/to/SRR925687_2.fastq path/to/human.refseq.gff path/to/RNA.test

I am getting error in this step (STEP4). Error message is as follows:

/data/ngs/algorithms/star/STAR/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 2  --genomeLoad NoSharedMemory  --outSAMtype BAM Unsorted  --quantMode TranscriptomeSAM  --outSAMheaderHD \@HD VN:1.4 SO:unsorted  --outFileNamePrefix RNA.test.temp/RNA.test  --readFilesIn SRR925687_1.fastq SRR925687_2.fastq sh: 1:
/data/ngs/algorithms/star/STAR/STAR: not found
"/data/ngs/algorithms/star/STAR/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 2  --genomeLoad NoSharedMemory  --outSAMtype BAM Unsorted  --quantMode TranscriptomeSAM  --outSAMheaderHD \@HD VN:1.4 SO:unsorted  --outFileNamePrefix RNA.test.temp/RNA.test  --readFilesIn SRR925687_1.fastq SRR925687_2.fastq" failed! Plase check if you
provide correct parameters/options for the pipeline!

STEP5-calculate expression without using SATR:

path/to/rsem-calculate-expression -p 2 --output-genome-bam --bowtie2
--bowtie2-path path/to/bowtie2 --paired-end path/to/SRR925687_1.fastq path/to/SRR925687_2.fastq human.refseq.gff path/to/RNA.run

I am getting error in this step too (STEP5). Error message is as follows:

path /data/ngs/algorithms/bowtie2 --paired-end SRR925687_1.fastq
SRR925687_2.fastq human.refseq.gff RNA.run sudo: unable to resolve
host dev.iphronesis.local /data/ngs/algorithms/bowtie2/bowtie2 -q
--phred33 --sensitive --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.gff -1 SRR925687_1.fastq -2 SRR925687_2.fastq |
samtools view -S -b -o RNA.run.temp/RNA.run.bam - samtools:
/lib/x86_64-linux-gnu/libm.so.6: version `GLIBC_2.23' not found
(required by samtools) (ERR): bowtie2-align exited with value 141
"/data/ngs/algorithms/bowtie2/bowtie2 -q --phred33 --sensitive --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.gff -1 SRR925687_1.fastq -2 SRR925687_2.fastq | samtools view -S -b -o
RNA.run.temp/RNA.run.bam -" failed! Plase check if you provide correct
parameters/options for the pipeline!

Also, I want to check for rsem version. I tried the following commands:

path/to/rsem --version 
path/to/rsem --help
rsem-calculate-expression  --help

I got following message:

rsem: command not found

I tried with all possible combinations to run the above pipeline successfully, but cant. Kindly, help.

Also, spare me for being so elaborative.

ADD COMMENTlink modified 17 months ago by RamRS24k • written 18 months ago by shuksi198450

For the Step 4 error, check that /data/ngs/algorithms/star/STAR/STAR is actually executable.

For the Step 5 error, ensure that samtools is in your PATH variable, and also update your libc6 libraries:

sudo apt-get update
sudo apt-get install libc6
ADD REPLYlink modified 18 months ago • written 18 months ago by Kevin Blighe50k

Following files are there in star directory:

drwxrwxr-x  7 stuser stuser    4096 Feb 18 08:47 ./
drwxrwxr-x 33 stuser stuser    4096 Apr  2 15:11 ../
drwxrwxr-x  5 stuser stuser    4096 Feb 15 14:25 bin/
-rwxrwxr-x  1 stuser stuser   21137 Feb 18 08:53 CHANGES.md*
drwxrwxr-x  2 stuser stuser    4096 Feb 15 14:32 doc/
drwxrwxr-x  4 stuser stuser    4096 Feb 15 14:21 extras/
drwxrwxr-x  8 stuser stuser    4096 Feb 15 14:32 .git/
-rwxrwxr-x  1 stuser stuser     210 Feb 18 08:53 .gitignore*
-rwxrwxr-x  1 stuser stuser       0 Feb 18 08:53 .gitmodules*
-rwxrwxr-x  1 stuser stuser     667 Feb 18 08:53 LICENSE*
-rwxrwxr-x  1 stuser stuser    3251 Feb 18 08:53 README.md*
-rwxrwxr-x  1 stuser stuser    4921 Feb 18 08:53 RELEASEnotes.md*
drwxrwxr-x  3 stuser stuser   12288 Feb 15 14:25 source/
-rwxr-xr-x  1 stuser stuser 2250176 Feb 18 08:53 star*
-rwxrwxr-x  1 stuser stuser 2250176 Feb 18 08:53 STAR*
-rwxrwxr-x  1 stuser stuser     273 Feb 18 08:53 .travis.yml*

All has execute permission

ADD REPLYlink modified 17 months ago by RamRS24k • written 17 months ago by shuksi198450

Did you install libc6 as instructed?

ADD REPLYlink written 17 months ago by Kevin Blighe50k

Yes, I did but theres mistake in STEP -3

path/to/STAR --runThreadN 2 --runMode
genomeGenerate --genomeDir /path/to/genome_file --genomeFastaFiles
path/to/GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna
--sjdbGTFfile path/to/human.refseq.gff --sjdbOverhang 100 --limitGenomeGenerateRAM=8825541333 --genomeSAsparseD 2

Instead of GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna file GCF_000001405.31_GRCh38.p5_genomic.fna must be used for --genomeFastaFiles.

ADD REPLYlink modified 17 months ago • written 17 months ago by shuksi198450

Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.
code_formatting

Do not use the > (quote) option - that is not the right one for code.

ADD REPLYlink written 17 months ago by RamRS24k

Thank you for correcting me. Will follow the same

ADD REPLYlink written 17 months ago by shuksi198450
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