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I am trying to run STAR alignment for pair-end reads and keep getting this error: "EXITING: because of fatal INPUT file error: could not open read file: Read1. I can't find where is the problem in my syntax. Here is my code:

cd /cs/labs/michall/yaelh/Projects/dsRNA-seq

# Mapping to masked genome:
STAR --runThreadN 6  \
     --runMode alignReads \
     --readFilesCommand gunzip -c \
     --genomeDir /Star_indices/Nve_genome_masked_index \     
     --readFilesIn  /output_files/Trimmomatic_out_paired/ad1_IgG_S10_R1_001_Trimmo_out_paired.fastq.gz  \
                    /output_files/Trimmomatic_out_paired/ad1_IgG_S10_R3_001_Trimmo_out_paired.fastq.gz  \       
     --outFileNamePrefix /Star_results/ad1_IgG_S10_R1_ \
     --outSAMtype BAM SortedByCoordinate \
     --outSAMunmapped Within \
     --outSAMattributes Standard \
     --quantMode TrancriptomSam GeneCount
Marcus Müller
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Yael H
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  • Is the genomeDir `/cs/labs/michall/yaelh/Projects/dsRNA-seq/Star_indices/Nve_genome_masked_index` or `/Star_indices/Nve_genome_masked_index`? – Fravadona Jan 03 '22 at 13:33
  • The option `--readFilesCommand gunzip -c` may also have to be replaced by `--readFilesCommand 'gunzip -c'` but I'm not sure, as I don't know the STAR command – Fravadona Jan 03 '22 at 13:36
  • The full path to the index file is: /cs/labs/michall/yaelh/Projects/dsRNA-seq/Star_indices/Nve_genome_masked_index – Yael H Jan 03 '22 at 13:42
  • OK, then remove all the prefixing `/` in the STAR command because they imply a full path. Example: `--genomeDir /Star_indices/Nve_genome_masked_index` has to be changed to `--genomeDir Star_indices/Nve_genome_masked_index` – Fravadona Jan 03 '22 at 13:43
  • according to STAR manual it's : --readFilesCommand gunzip -c (without quotation marks) – Yael H Jan 03 '22 at 13:45
  • https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf seems to be saying that the argument to `--readFilesIn` should be a single string with a comma between the file names. (That's a horrible design, if it's true.) – tripleee Jan 03 '22 at 13:47
  • I tried. It didn't help :( – Yael H Jan 03 '22 at 13:48
  • a comma is used between multiple samples but not between a paired end – Yael H Jan 03 '22 at 13:49

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