rsem-calculate-expression error message

Question

[shuksi1984]

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.

[Kevin Blighe]

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

[shuksi1984]

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

[Kevin Blighe]

Did you install libc6 as instructed?

[shuksi1984]

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.

[Ram]

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

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

[shuksi1984]

Thank you for correcting me. Will follow the same