Answers.Body: <p>I believe the error is from <code>featureCounts</code> and the solution would be to add the <code>-p</code> flag to the <code>featureCounts</code> call, stating that you are providing paired-end data.</p>
  <p>EDIT: You might want to check this <a href="https://www.reddit.com/r/bioinformatics/comments/13m89bd/help_with_featurecounts_error_pairedend_reads/" rel="noreferrer"><code>r/bioinformatics</code> (reddit) thread</a> out.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-12-23T05:41:00.363Z
Answers.Text: Well, thanks. It worked when I added the -p flag to the featureCounts call
Answers.UserDisplayName: Emmanuel Aroma
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Answers.CreationDate: 2023-12-24T16:16:19.773Z
Answers.Text: @EmmanuelAroma Accept haci's answer to mark the post as answered.
Answers.UserDisplayName: Ram RS
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-12-25T07:03:31.523Z
Answers.Text: thank you so much comrades, I've got a wonderful answer
Answers.UserDisplayName: Emmanuel Aroma
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-12-22T13:28:26.453Z
Answers.LastActivityDate: 2023-12-22T13:28:26.453Z
Answers.UserDisplayName: haci
Body: <p><em>This question was also asked on <a href="https://www.biostars.org/p/9583255/" rel="nofollow noreferrer">Biostars</a></em></p>
  <p>I have this script</p>
  <pre><code>#!/bin/bash
  
  # Defining the number of threads to be used
  threads=20
  
  # Alignment of sequences to the reference genome using 'HISAT2'
  echo -e &quot;\nNow Running Classical Alignment-based with HISAT2...\n&quot;
  
  mkdir -p hisat2
  
  # Download the human reference genome
  wget -c ftp://ftp.ensembl.org/pub/release-100/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz -O hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
  gunzip hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
  
  # Download the human annotation file
  wget -c ftp://ftp.ensembl.org/pub/release-100/gtf/homo_sapiens/Homo_sapiens.GRCh38.100.gtf.gz -O hisat2/Homo_sapiens.GRCh38.100.gtf.gz
  gunzip hisat2/Homo_sapiens.GRCh38.100.gtf.gz
  
  # HISAT2 indexing to build the reference genome index
  hisat2-build -p $threads hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa hisat2/Homo_sapiens.GRCh38v3_hisat2.idx
  
  # HISAT2 Alignment
  for sample in <span class="math-container">$(cat sample_ids.txt)
  do
      hisat2 -p $</span>threads -x hisat2/Homo_sapiens.GRCh38v3_hisat2.idx \
          -1 trimmed_data/<span class="math-container">${sample}_1_val_1.fq -2 trimmed_data/$</span>{sample}_2_val_2.fq \
          -S hisat2/${sample}_hisat2.sam
  
      # Compress the SAM files to binary format, sort them, and index them.
      samtools view -Sb hisat2/<span class="math-container">${sample}_hisat2.sam | samtools sort &gt; hisat2/$</span>{sample}_hisat2_sorted.bam
      samtools index hisat2/<span class="math-container">${sample}_hisat2_sorted.bam
  rm hisat2/$</span>{sample}_hisat2.sam  # Remove the .sam files for storage purposes
  done
  
  # Quantifying Aligned reads using the Subread package's script 'featureCounts'
  for sample in <span class="math-container">$(cat sample_ids.txt)
  do
  featureCounts -T $</span>threads -a hisat2/Homo_sapiens.GRCh38.100.gtf \
          -o hisat2/<span class="math-container">${sample}_hisat2_counts.txt \
      hisat2/$</span>{sample}_hisat2_sorted.bam
  done
  
  # Generating analysis report with MultiQC
  echo &quot;Running MultiQC on HISAT2 alignment results .........&quot;
  multiqc -o multiQCreports hisat2
  echo &quot;MultiQC on HISAT2 alignment results completed successfully.&quot;
  
  # Display message to show that the script is complete
  echo &quot;Script completed!&quot;
  </code></pre>
  <p>However, I have receieved an error below</p>
  <pre><code>Load annotation file Homo_sapiens.GRCh38.100.gtf ...                       ||
  ||    Features : 1378275                                                      ||
  ||    Meta-features : 60683                                                   ||
  ||    Chromosomes/contigs : 47                                                ||
  ||                                                                            ||
  || Process BAM file SRR19170514_hisat2_sorted.bam...                          ||
  ERROR: Paired-end reads were detected in single-end read library : hisat2/SRR19170514_hisat2_sorted.bam
  ERROR: Paired-end reads were detected in single-end read library : hisat2/SRR19170514_hisat2_sorted.bam
  </code></pre>
  <p>Question: What can I do to solve this?</p>
  
Tags: <rna-seq>
Title: Classical alignment with HISAT2
ThreadText: # Classical alignment with HISAT2
  
  (Asked by: username_0 on 2023-12-22)
  
  *This question was also asked on [Biostars](https://www.biostars.org/p/9583255/)*
  
  I have this script
  
  ```
  #!/bin/bash
  
  # Defining the number of threads to be used
  threads=20
  
  # Alignment of sequences to the reference genome using 'HISAT2'
  echo -e "\nNow Running Classical Alignment-based with HISAT2...\n"
  
  mkdir -p hisat2
  
  # Download the human reference genome
  wget -c ftp://ftp.ensembl.org/pub/release-100/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz -O hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
  gunzip hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
  
  # Download the human annotation file
  wget -c ftp://ftp.ensembl.org/pub/release-100/gtf/homo_sapiens/Homo_sapiens.GRCh38.100.gtf.gz -O hisat2/Homo_sapiens.GRCh38.100.gtf.gz
  gunzip hisat2/Homo_sapiens.GRCh38.100.gtf.gz
  
  # HISAT2 indexing to build the reference genome index
  hisat2-build -p $threads hisat2/Homo_sapiens.GRCh38.dna.primary_assembly.fa hisat2/Homo_sapiens.GRCh38v3_hisat2.idx
  
  # HISAT2 Alignment
  for sample in $(cat sample_ids.txt)
  do
      hisat2 -p $threads -x hisat2/Homo_sapiens.GRCh38v3_hisat2.idx \
          -1 trimmed_data/${sample}_1_val_1.fq -2 trimmed_data/${sample}_2_val_2.fq \
          -S hisat2/${sample}_hisat2.sam
  
      # Compress the SAM files to binary format, sort them, and index them.
      samtools view -Sb hisat2/${sample}_hisat2.sam | samtools sort > hisat2/${sample}_hisat2_sorted.bam
      samtools index hisat2/${sample}_hisat2_sorted.bam
  rm hisat2/${sample}_hisat2.sam  # Remove the .sam files for storage purposes
  done
  
  # Quantifying Aligned reads using the Subread package's script 'featureCounts'
  for sample in $(cat sample_ids.txt)
  do
  featureCounts -T $threads -a hisat2/Homo_sapiens.GRCh38.100.gtf \
          -o hisat2/${sample}_hisat2_counts.txt \
      hisat2/${sample}_hisat2_sorted.bam
  done
  
  # Generating analysis report with MultiQC
  echo "Running MultiQC on HISAT2 alignment results ........."
  multiqc -o multiQCreports hisat2
  echo "MultiQC on HISAT2 alignment results completed successfully."
  
  # Display message to show that the script is complete
  echo "Script completed!"
  
  ```
  
  However, I have receieved an error below
  
  ```
  Load annotation file Homo_sapiens.GRCh38.100.gtf ...                       ||
  ||    Features : 1378275                                                      ||
  ||    Meta-features : 60683                                                   ||
  ||    Chromosomes/contigs : 47                                                ||
  ||                                                                            ||
  || Process BAM file SRR19170514_hisat2_sorted.bam...                          ||
  ERROR: Paired-end reads were detected in single-end read library : hisat2/SRR19170514_hisat2_sorted.bam
  ERROR: Paired-end reads were detected in single-end read library : hisat2/SRR19170514_hisat2_sorted.bam
  
  ```
  
  Question: What can I do to solve this?
  
  **Tags:** rna-seq
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2023-12-22. Score: 6 | Accepted answer
  
  I believe the error is from `featureCounts` and the solution would be to add the `-p` flag to the `featureCounts` call, stating that you are providing paired-end data.
  
  EDIT: You might want to check this [`r/bioinformatics` (reddit) thread](https://www.reddit.com/r/bioinformatics/comments/13m89bd/help_with_featurecounts_error_pairedend_reads/) out.
  
  > **Comments:**
  > **username_0**: Well, thanks. It worked when I added the -p flag to the featureCounts call  
  > **username_2**: @username_0 Accept username_1's answer to mark the post as answered.  
  > **username_0**: thank you so much comrades, I've got a wonderful answer  
  
  ---
  
  