Body: <p>I have mapped reads to the yeast (R64-1-1) genome with STAR like this:</p>
  <pre><code>STAR --runThreadN 44 --runMode alignReads --genomeDir /home/user/Documents/scripts/pyseqtools/index/STAR/R64-1-1 --readFilesIn /home/user/Documents/analyses/TT-Seq/user/trim/WT_3_R1_001_val_1.fq.gz /home/user/Documents/analyses/TT-Seq/user/trim/WT_3_R2_001_val_2.fq.gz --readFilesCommand zcat --quantMode TranscriptomeSAM GeneCounts --twopassMode Basic --outSAMunmapped None --outSAMattrRGline ID:WT_3 PU:WT_3 SM:WT_3 LB:unknown PL:illumina --outSAMtype BAM Unsorted --outTmpDir /home/user/Documents/analyses/TT-Seq/user/tmp --outFileNamePrefix /home/user/Documents/analyses/TT-Seq/user/bam/R64-1-1/WT_3/WT_3
  </code></pre>
  <p>I wanted to count the reads using <code>htseq-count</code> as follows:</p>
  <pre><code>htseq-count --quiet --format bam --stranded yes /home/user/Documents/analyses/TT-Seq/user/bam/R64-1-1/WT_3/WT_3Aligned.out.bam /home/user/Documents/scripts/pyseqtools/gtf/R64-1-1/Saccharomyces_cerevisiae.R64-1-1.106.gtf &gt; /home/user/Documents/analyses/TT-Seq/user/htseq-count/WT_3.count.txt
  </code></pre>
  <p>The problem is that most reads map to <code>no_feature</code>:</p>
  <pre><code>__no_feature    4462786
  __ambiguous 12644
  __too_low_aQual 0
  __not_aligned   4907
  __alignment_not_unique  12529444
  </code></pre>
  <p>I have check whether the chromosome names in the BAM file and the GTF file matched (which they seem to do):</p>
  <pre><code>BAM:
  @HD VN:1.4
  @SQ SN:I    LN:230218
  @SQ SN:II   LN:813184
  @SQ SN:III  LN:316620
  @SQ SN:IV   LN:1531933
  @SQ SN:V    LN:576874
  @SQ SN:VI   LN:270161
  @SQ SN:VII  LN:1090940
  @SQ SN:VIII LN:562643
  @SQ SN:IX   LN:439888
  @SQ SN:X    LN:745751
  @SQ SN:XI   LN:666816
  @SQ SN:XII  LN:1078177
  @SQ SN:XIII LN:924431
  @SQ SN:XIV  LN:784333
  @SQ SN:XV   LN:1091291
  @SQ SN:XVI  LN:948066
  @SQ SN:Mito LN:85779
  
  GTF: 
  IV  sgd gene    8683    9756    .   -   .   gene_id &quot;YDL246C&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;;
  IV  sgd transcript  8683    9756    .   -   .   gene_id &quot;YDL246C&quot;; transcript_id &quot;YDL246C_mRNA&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;SOR2&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd exon    8683    9756    .   -   .   gene_id &quot;YDL246C&quot;; transcript_id &quot;YDL246C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;SOR2&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; exon_id &quot;YDL246C_mRNA-E1&quot;;
  IV  sgd CDS 8686    9756    .   -   0   gene_id &quot;YDL246C&quot;; transcript_id &quot;YDL246C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;SOR2&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; protein_id &quot;YDL246C&quot;;
  IV  sgd start_codon 9754    9756    .   -   0   gene_id &quot;YDL246C&quot;; transcript_id &quot;YDL246C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;SOR2&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd stop_codon  8683    8685    .   -   0   gene_id &quot;YDL246C&quot;; transcript_id &quot;YDL246C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;SOR2&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;SOR2&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd gene    17577   18566   .   -   .   gene_id &quot;YDL243C&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;;
  IV  sgd transcript  17577   18566   .   -   .   gene_id &quot;YDL243C&quot;; transcript_id &quot;YDL243C_mRNA&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;AAD4&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd exon    17577   18566   .   -   .   gene_id &quot;YDL243C&quot;; transcript_id &quot;YDL243C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;AAD4&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; exon_id &quot;YDL243C_mRNA-E1&quot;;
  IV  sgd CDS 17580   18566   .   -   0   gene_id &quot;YDL243C&quot;; transcript_id &quot;YDL243C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;AAD4&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; protein_id &quot;YDL243C&quot;;
  IV  sgd start_codon 18564   18566   .   -   0   gene_id &quot;YDL243C&quot;; transcript_id &quot;YDL243C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;AAD4&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd stop_codon  17577   17579   .   -   0   gene_id &quot;YDL243C&quot;; transcript_id &quot;YDL243C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;AAD4&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;AAD4&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd gene    1248154 1249821 .   -   .   gene_id &quot;YDR387C&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;;
  IV  sgd transcript  1248154 1249821 .   -   .   gene_id &quot;YDR387C&quot;; transcript_id &quot;YDR387C_mRNA&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;CIN10&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd exon    1248154 1249821 .   -   .   gene_id &quot;YDR387C&quot;; transcript_id &quot;YDR387C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;CIN10&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; exon_id &quot;YDR387C_mRNA-E1&quot;;
  IV  sgd CDS 1248157 1249821 .   -   0   gene_id &quot;YDR387C&quot;; transcript_id &quot;YDR387C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;CIN10&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;; protein_id &quot;YDR387C&quot;;
  IV  sgd start_codon 1249819 1249821 .   -   0   gene_id &quot;YDR387C&quot;; transcript_id &quot;YDR387C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;CIN10&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  IV  sgd stop_codon  1248154 1248156 .   -   0   gene_id &quot;YDR387C&quot;; transcript_id &quot;YDR387C_mRNA&quot;; exon_number &quot;1&quot;; gene_name &quot;CIN10&quot;; gene_source &quot;sgd&quot;; gene_biotype &quot;protein_coding&quot;; transcript_name &quot;CIN10&quot;; transcript_source &quot;sgd&quot;; transcript_biotype &quot;protein_coding&quot;;
  
  
  </code></pre>
  <p>Edit: the output of <code>ReadsPerGene.out.tab</code> is (thanks
  swbarnes2) :</p>
  <pre><code>N_unmapped  73359652    73359652    73359652
  N_multimapping  15789900    15789900    15789900
  N_noFeature 192619  5872967 380420
  N_ambiguous 648956  13479   430527
  </code></pre>
  <p>Would anyone know why most reads map to <code>no_feature</code>?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2022-07-27T22:16:08.550Z
Comments.Text: You are already running it with GeneCounts, which should output the same as htseq-count.  What does your ReadsPerGene.out.tab say?
Comments.UserDisplayName: swbarnes2
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2022-07-29T11:18:09.333Z
Comments.Text: Do your gtf contains exon entris (i.e. with "exon" on the 3rd column)? Could you please update your question with a few exon entries from your gtf?
Comments.UserDisplayName: bepoli
Tags: <phylogenetics><star>
Title: Most reads no_feature with htseq-count
ThreadText: # Most reads no_feature with htseq-count
  
  (Asked by: username_0 on 2022-07-26)
  
  I have mapped reads to the yeast (R64-1-1) genome with STAR like this:
  
  ```
  STAR --runThreadN 44 --runMode alignReads --genomeDir /home/user/Documents/scripts/pyseqtools/index/STAR/R64-1-1 --readFilesIn /home/user/Documents/analyses/TT-Seq/user/trim/WT_3_R1_001_val_1.fq.gz /home/user/Documents/analyses/TT-Seq/user/trim/WT_3_R2_001_val_2.fq.gz --readFilesCommand zcat --quantMode TranscriptomeSAM GeneCounts --twopassMode Basic --outSAMunmapped None --outSAMattrRGline ID:WT_3 PU:WT_3 SM:WT_3 LB:unknown PL:illumina --outSAMtype BAM Unsorted --outTmpDir /home/user/Documents/analyses/TT-Seq/user/tmp --outFileNamePrefix /home/user/Documents/analyses/TT-Seq/user/bam/R64-1-1/WT_3/WT_3
  
  ```
  
  I wanted to count the reads using `htseq-count` as follows:
  
  ```
  htseq-count --quiet --format bam --stranded yes /home/user/Documents/analyses/TT-Seq/user/bam/R64-1-1/WT_3/WT_3Aligned.out.bam /home/user/Documents/scripts/pyseqtools/gtf/R64-1-1/Saccharomyces_cerevisiae.R64-1-1.106.gtf > /home/user/Documents/analyses/TT-Seq/user/htseq-count/WT_3.count.txt
  
  ```
  
  The problem is that most reads map to `no_feature`:
  
  ```
  __no_feature    4462786
  __ambiguous 12644
  __too_low_aQual 0
  __not_aligned   4907
  __alignment_not_unique  12529444
  
  ```
  
  I have check whether the chromosome names in the BAM file and the GTF file matched (which they seem to do):
  
  ```
  BAM:
  @HD VN:1.4
  @SQ SN:I    LN:230218
  @SQ SN:II   LN:813184
  @SQ SN:III  LN:316620
  @SQ SN:IV   LN:1531933
  @SQ SN:V    LN:576874
  @SQ SN:VI   LN:270161
  @SQ SN:VII  LN:1090940
  @SQ SN:VIII LN:562643
  @SQ SN:IX   LN:439888
  @SQ SN:X    LN:745751
  @SQ SN:XI   LN:666816
  @SQ SN:XII  LN:1078177
  @SQ SN:XIII LN:924431
  @SQ SN:XIV  LN:784333
  @SQ SN:XV   LN:1091291
  @SQ SN:XVI  LN:948066
  @SQ SN:Mito LN:85779
  
  GTF: 
  IV  sgd gene    8683    9756    .   -   .   gene_id "YDL246C"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding";
  IV  sgd transcript  8683    9756    .   -   .   gene_id "YDL246C"; transcript_id "YDL246C_mRNA"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "SOR2"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd exon    8683    9756    .   -   .   gene_id "YDL246C"; transcript_id "YDL246C_mRNA"; exon_number "1"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "SOR2"; transcript_source "sgd"; transcript_biotype "protein_coding"; exon_id "YDL246C_mRNA-E1";
  IV  sgd CDS 8686    9756    .   -   0   gene_id "YDL246C"; transcript_id "YDL246C_mRNA"; exon_number "1"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "SOR2"; transcript_source "sgd"; transcript_biotype "protein_coding"; protein_id "YDL246C";
  IV  sgd start_codon 9754    9756    .   -   0   gene_id "YDL246C"; transcript_id "YDL246C_mRNA"; exon_number "1"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "SOR2"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd stop_codon  8683    8685    .   -   0   gene_id "YDL246C"; transcript_id "YDL246C_mRNA"; exon_number "1"; gene_name "SOR2"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "SOR2"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd gene    17577   18566   .   -   .   gene_id "YDL243C"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding";
  IV  sgd transcript  17577   18566   .   -   .   gene_id "YDL243C"; transcript_id "YDL243C_mRNA"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "AAD4"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd exon    17577   18566   .   -   .   gene_id "YDL243C"; transcript_id "YDL243C_mRNA"; exon_number "1"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "AAD4"; transcript_source "sgd"; transcript_biotype "protein_coding"; exon_id "YDL243C_mRNA-E1";
  IV  sgd CDS 17580   18566   .   -   0   gene_id "YDL243C"; transcript_id "YDL243C_mRNA"; exon_number "1"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "AAD4"; transcript_source "sgd"; transcript_biotype "protein_coding"; protein_id "YDL243C";
  IV  sgd start_codon 18564   18566   .   -   0   gene_id "YDL243C"; transcript_id "YDL243C_mRNA"; exon_number "1"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "AAD4"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd stop_codon  17577   17579   .   -   0   gene_id "YDL243C"; transcript_id "YDL243C_mRNA"; exon_number "1"; gene_name "AAD4"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "AAD4"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd gene    1248154 1249821 .   -   .   gene_id "YDR387C"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding";
  IV  sgd transcript  1248154 1249821 .   -   .   gene_id "YDR387C"; transcript_id "YDR387C_mRNA"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "CIN10"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd exon    1248154 1249821 .   -   .   gene_id "YDR387C"; transcript_id "YDR387C_mRNA"; exon_number "1"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "CIN10"; transcript_source "sgd"; transcript_biotype "protein_coding"; exon_id "YDR387C_mRNA-E1";
  IV  sgd CDS 1248157 1249821 .   -   0   gene_id "YDR387C"; transcript_id "YDR387C_mRNA"; exon_number "1"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "CIN10"; transcript_source "sgd"; transcript_biotype "protein_coding"; protein_id "YDR387C";
  IV  sgd start_codon 1249819 1249821 .   -   0   gene_id "YDR387C"; transcript_id "YDR387C_mRNA"; exon_number "1"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "CIN10"; transcript_source "sgd"; transcript_biotype "protein_coding";
  IV  sgd stop_codon  1248154 1248156 .   -   0   gene_id "YDR387C"; transcript_id "YDR387C_mRNA"; exon_number "1"; gene_name "CIN10"; gene_source "sgd"; gene_biotype "protein_coding"; transcript_name "CIN10"; transcript_source "sgd"; transcript_biotype "protein_coding";
  
  ```
  
  Edit: the output of `ReadsPerGene.out.tab` is (thanks
  username_1) :
  
  ```
  N_unmapped  73359652    73359652    73359652
  N_multimapping  15789900    15789900    15789900
  N_noFeature 192619  5872967 380420
  N_ambiguous 648956  13479   430527
  
  ```
  
  Would anyone know why most reads map to `no_feature`?
  
  **Tags:** phylogenetics, star
  
  > **Comments:**
  > **username_1**: You are already running it with GeneCounts, which should output the same as htseq-count.  What does your ReadsPerGene.out.tab say?  
  > **username_2**: Do your gtf contains exon entris (i.e. with "exon" on the 3rd column)? Could you please update your question with a few exon entries from your gtf?  
  
  ---
  
  ## 0 answers
  
  