For loop RNA Sequencing alignment

Question

[kcarey]

Hey all, I was wondering if you guys could help me figure out how to correct the "--ReadFilesIn" part of my code....I keep getting "EXITING because of fatal input ERROR: could not open readFilesIn=.{sample}.fastq"

This is the code I used:

#Aligning all fastq files to HG-38
for i in cat MatchedFastqList.txt | while read samples do 
  STAR --genomeDir /projects/home/genomeDir --runThreadN 16 --runMode alignReads --readFilesIn ${sample}.{sample}.fastq --sjdbGTFfile /projects/home/genomeDir 19/GCF_000001405.40_GRCh38.p14_genomic.gtf --outReadsUnmapped Fastx done

Also....this is the fastq formats in my list MatchedFastqList.txt " OV33.FBHHHHHHS_G7_ITGACVA.fastq"

[iraun]

Try something like this:

while read f1; do
    STAR ... --readFilesIn "$f1"
done<MatchedFastqList.txt

[kcarey]

Hey! Thank you so much for that. I tried that! Still getting error

EXITING because of fatal input ERROR: could not open readFilesIn=Read1

[iraun]

Hmm.. Are the file names in MatchedFastqList.txt with quotes?

Also, have you tried to just pick the first line from MatchedFastqList.txt and try to run STAR (without the loop)?

[Medhat]

Can you show us an example of what MatchedFastqList.txt looks like? E.g., head MatchedFastqList.txt

[kcarey]

I have discovered it's not the loop, it is correctly grabbing the files, the issue is it's telling me it can not read the GenomeDir.

EXITING because of FATAL ERROR: could not open genome file /projects/home/kcarey8/FinalMayodownloads/Matched/Batch2/Fastq/Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna//genomeParameters.txt SOLUTION: check that the path to genome files, specified in --genomeDir is correct and the files are present, and have user read permsissions

Oct 17 10:51:28 ...... FATAL ERROR, exiting STAR --genomeDir /Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna --runThreadN 20 --runMode alignReads --readFilesIn OV74.FCD1U68ACXX_L8_IACTGAT.fastq --sjdbGTFfile /Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.gtf --outSAMtype BAM Unsorted --outReadsUnmapped Fastx --outFileNamePrefix Aligned_OV74.FCD1U68ACXX_L8_IACTGAT.fastq STAR version: 2.7.10a compiled: 2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Oct 17 10:51:28 ..... started STAR run Oct 17 10:51:28 ..... loading genome

I have also Chmod 755 all files so they are executable. For some reason it does not like the code....I used the absolute file path below:

for i in *.fastq ; do STAR --genomeDir /Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna --runThreadN 20 --runMode alignReads --readFilesIn ${i} --sjdbGTFfile /Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.gtf --outSAMtype BAM Unsorted --outReadsUnmapped Fastx --outFileNamePrefix Aligned_${i} ; done

[iraun]

You are reporting different errors.

First, in the original question, you specified the error was: "EXITING because of fatal input ERROR: could not open readFilesIn=.{sample}.fastq"

Now, you are saying the error is: "EXITING because of FATAL ERROR: could not open genome file"

In addition, it seems you are mixing the paths and the files, f.e:

• /projects/home/kcarey8/FinalMayodownloads/Matched/Batch2/Fastq/Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna//genomeParameters.txt

genomeParameters.txt should not be the "genome file"

• /Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna
• /projects/home/kcarey8/FinalMayodownloads/Matched/Batch2/Fastq/Indexes/ncbi-genomes-2022-09-19/GCF_000001405.40_GRCh38.p14_genomic.fna

I suggest to clean up a bit your code, and command, and report the exact error you are getting.

[kcarey]

As I fix the issues I develop a new issue. I am not trying to confuse the errors.