Entering edit mode
6.1 years ago
shuksi1984
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60
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.
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:
Following files are there in star directory:
All has execute permission
Did you install
libc6
as instructed?Yes, I did but theres mistake in STEP -3
Instead of GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna file GCF_000001405.31_GRCh38.p5_genomic.fna must be used for --genomeFastaFiles.
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.Thank you for correcting me. Will follow the same