MARS seq alingment

Question

[roniozagam94]

Hello everyone, new here and also new to the field. was asked to create a pipeline for RNA seq and after two months of self learning of how to interact with each code im stuck with the program STAR. what im trying to do for now is genomegenerate, ill add the code that i wrote here and the problem. hope anyone can help me. tried to write "sudo" before the command and it didnt work. NOTE: im not familiar with these languages, undergraduate in biomed studies so be patient if you may.. hehe

the code:

 STAR --runThreadN 6\
 --runMode genomeGenerate \
 --genomeDir ~/ncbi/public/sra/testercut \
 --genomeFastaFiles /ncbi/public/sra/testercut/GCA_009858895.3.fasta \
 --sjdbGTFfile /ncbi/public/sra/testercut/COVID_19_gene_annotation.gtf \
 --outFileNamePrefix /ncbi/public/sra/testercut/STARGEN \
 --sjdbOverhang 99

EXITING because of fatal OUTPUT FILE error: could not create output directory: /ncbi for --outFileNamePrefix /ncbi/public/sra/testercut/STARGEN
 ERROR: Permission denied
SOLUTION: check the path and permissions.

Jan 05 09:59:42 ...... FATAL ERROR, exiting

[ATpoint]

/ncbi would mean there was a ncbi folder in root. You probably forgot the tilde as used in the genomeDir argument.

[rpolicastro]

I don't think --outFileNamePrefix is used during genome indexing, so can probably be removed.

[roniozagam94]

got it. now i got other problem after I removed the line of the prefix

STAR --runThreadN=6 \
 --runMode genomeGenerate \
 --genomeFastaFiles ~/ncbi/public/sra/testercut/GCA_009858895.3.fasta \
 --sjdbGTFfile /ncbi/public/sra/testercut/COVID19_gene_annotation.gtf \
 --sjdbOverhang 99

    STAR version: 2.7.9a   compiled: 2021-05-04T09:43:56-0400 vega:/home/dobin/data/STAR/STARcode/STAR.master/source
Jan 09 04:44:22 ..... started STAR run
Jan 09 04:44:22 ... starting to generate Genome files
Jan 09 04:44:22 ..... processing annotations GTF

FATAL error, could not open file pGe.sjdbGTFfile=/ncbi/public/sra/testercut/COVID19_gene_annotation.gtf

Jan 09 04:44:22 ...... FATAL ERROR, exiting

why is that?

[ATpoint]

Did you even read my comment? Your paths are wrong. You mix ~/ncbi an /ncbi. In all likelyhood, there is no filder /ncbi. Just use ls /ncbi, I bet it does not exist. Change the lines into ~/ncbi given that even exists. Do you understand my point, if not please tell how I should explain better.

[roniozagam94]

sorry about that, it worked. however now ill need to find a proper fasta/gtf file that correlate "Fatal INPUT FILE error, no valid exon lines in the GTF file: /home/tester/ncbi/public/sra/testercut/COVID19_gene_annotation.gtf Solution: check the formatting of the GTF file. One likely cause is the difference in chromosome naming between GTF and FASTA file."