Error in opening fastq.gz file

Question

[Jakpa]

Hello Everyone,

I want to perform Adapter trimming. After running this command,

flexbar --adapter-min-overlap 7 --adapter-trim-end RIGHT \
        --adapters ~/RNASeq/rawDataTrim/illumina_multiplex.fa \
        --pre-trim-left 13 --max-uncalled 300 --min-read-length 25 \
        --threads 2 --zip-output GZ \
        --reads ~/RNASeq/rawData/UHR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz \
        --reads2 ~/RNASeq/rawData/UHR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz \
        --target ~/RNASeq/rawDataTrim/UHR_1
flexbar --adapter-min-overlap 7 --adapter-trim-end RIGHT \
        --adapters ~/RNASeq/rawDataTrim/illumina_multiplex.fa \
        --pre-trim-left 13 --max-uncalled 300 --min-read-length 25 \
        --threads 2 --zip-output GZ \
        --reads ~/RNASeq/rawData/UHR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz \
        --reads2 ~/RNASeq/rawData/UHR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz \
        --target ~/RNASeq/rawDataTrim/UHR_2
flexbar --adapter-min-overlap 7 --adapter-trim-end RIGHT \
        --adapters ~/RNASeq/rawDataTrim/illumina_multiplex.fa \
        --pre-trim-left 13 --max-uncalled 300 --min-read-length 25 \
        --threads 2 --zip-output GZ \
        --reads ~/RNASeq/rawData/UHR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz \
        --reads2 ~/RNASeq/rawData/UHR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz \
        --target ~/RNASeq/rawDataTrim/UHR_3

I got this error:

ERROR: Could not open file /home/mlsi/RNASeq/rawData/UHR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz
ERROR: Could not open file /home/mlsi/RNASeq/rawData/UHR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz
ERROR: Could not open file /home/mlsi/RNASeq/rawData/UHR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz

When I opened my rawData dir, I saw that I have the files.

UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz
UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.html
UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.zip
UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz
UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.html
UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.zip
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.html
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.zip
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.html
UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.zip
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.html
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1_fastqc.zip
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.html
UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2_fastqc.zip

What am I doing wrong?

Regards,
Anthony

[Ram]

Please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or select a chunk of text and use the highlighted button to format it as a code block. If your code has long lines with a single command, break those lines into multiple lines with proper escape sequences so they're easier to read and still run when copy-pasted. I've done it for you this time.

P.S: Do not use the <pre> tag - it breaks the site's markdown based formatting.

[Jakpa]

Thank you for Educating me. Am new in this platform

noted henceforth

Regards,

Anthony

Answer 1

[Ram]

Your files have Mix1 in their names whereas your command uses Mix2. Unless I'm missing something, that needs to be fixed.

[Jakpa]

Thank you for spotting out my error. I have done the corrections and now everything is perfect...

This was the cause of my problem why I had duplicates reads in my samples in the FeatureCounts. The alignment was wrongly done .

Thank you all.. Anthony

[Ram]

If an answer was helpful, you should upvote it; if the answer resolved your question, you should mark it as accepted. You can accept more than one answer if they all work. If an answer was not really helpful or did not work, provide detailed feedback so others know not to use that answer.