text: user292: I have surprisingly low counts when running featureCounts on some (single-end) RNA-seq data mapped on C. elegans genome using hisat2.
  To more easily show the problem, I generated a small subset of the bam file and of the annotation file I'm using. Here is what I can see when loading these two files on IGV:
  
  The coverage for the "his-11" and "his-15" genes peak at around 3600. For "his-12" and "his-16", it peaks at more than 1000
  Here are the content of my annotation file:
  $ cat his_11-16.gtf 
  II  ensembl transcript  13817759    13818143    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001890"; gene_name "his-16"; gene_source "ensembl"; gene_version "1"; p_id "P8354"; transcript_biotype "protein_coding"; transcript_id "ZK131.10"; transcript_name "ZK131.10"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS11539";
  II  ensembl transcript  13818371    13818739    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001889"; gene_name "his-15"; gene_source "ensembl"; gene_version "1"; p_id "P8185"; transcript_biotype "protein_coding"; transcript_id "ZK131.9"; transcript_name "ZK131.9"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS52436";
  II  ensembl transcript  13819626    13819937    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001888"; gene_name "his-14"; gene_source "ensembl"; gene_version "1"; p_id "P21890"; transcript_biotype "protein_coding"; transcript_id "ZK131.8"; transcript_name "ZK131.8"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS50308";
  II  ensembl transcript  13820210    13820620    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001887"; gene_name "his-13"; gene_source "ensembl"; gene_version "1"; p_id "P2149"; transcript_biotype "protein_coding"; transcript_id "ZK131.7"; transcript_name "ZK131.7"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS35106";
  II  ensembl transcript  13821198    13821581    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001886"; gene_name "his-12"; gene_source "ensembl"; gene_version "1"; p_id "P26082"; transcript_biotype "protein_coding"; transcript_id "ZK131.6"; transcript_name "ZK131.6"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS23693";
  II  ensembl transcript  13821778    13822314    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001885"; gene_name "his-11"; gene_source "ensembl"; gene_version "1"; p_id "P10535"; transcript_biotype "protein_coding"; transcript_id "ZK131.5"; transcript_name "ZK131.5"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS52792";
  
  The featureCounts run:
  $ featureCounts -a his_11-16.gtf -o his_11-16_counts.txt -t transcript -g gene_name -O his_11-16_sorted.bam
  
          ==========     _____ _    _ ____  _____  ______          _____  
          =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
            =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
              ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
                ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
          ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
        v1.5.2
  
  //========================== featureCounts setting ===========================\\
  ||                                                                            ||
  ||             Input files : 1 BAM file                                       ||
  ||                           S his_11-16_sorted.bam                           ||
  ||                                                                            ||
  ||             Output file : his_11-16_counts.txt                             ||
  ||                 Summary : his_11-16_counts.txt.summary                     ||
  ||              Annotation : his_11-16.gtf (GTF)                              ||
  ||      Dir for temp files : ./                                               ||
  ||                                                                            ||
  ||                 Threads : 1                                                ||
  ||                   Level : meta-feature level                               ||
  ||              Paired-end : no                                               ||
  ||         Strand specific : no                                               ||
  ||      Multimapping reads : not counted                                      ||
  || Multi-overlapping reads : counted                                          ||
  ||   Min overlapping bases : 1                                                ||
  ||                                                                            ||
  \\===================== http://subread.sourceforge.net/ ======================//
  
  //================================= Running ==================================\\
  ||                                                                            ||
  || Load annotation file his_11-16.gtf ...                                     ||
  ||    Features : 6                                                            ||
  ||    Meta-features : 6                                                       ||
  ||    Chromosomes/contigs : 1                                                 ||
  ||                                                                            ||
  || Process BAM file his_11-16_sorted.bam...                                   ||
  ||    Single-end reads are included.                                          ||
  ||    Assign reads to features...                                             ||
  ||    Total reads : 35037                                                     ||
  ||    Successfully assigned reads : 1849 (5.3%)                               ||
  ||    Running time : 0.00 minutes                                             ||
  ||                                                                            ||
  ||                         Read assignment finished.                          ||
  ||                                                                            ||
  || Summary of counting results can be found in file "his_11-16_counts.txt"    ||
  ||                                                                            ||
  \\===================== http://subread.sourceforge.net/ ======================//
  
  And the resulting counts file:
  $ cat his_11-16_counts.txt
  # Program:featureCounts v1.5.2; Command:"featureCounts" "-a" "his_11-16.gtf" "-o" "his_11-16_counts.txt" "-t" "transcript" "-g" "gene_name" "-O" "his_11-16_sorted.bam" 
  Geneid  Chr Start   End Strand  Length  his_11-16_sorted.bam
  his-16  II  13817759    13818143    -   385 869
  his-15  II  13818371    13818739    +   369 3
  his-14  II  13819626    13819937    -   312 953
  his-13  II  13820210    13820620    +   411 23
  his-12  II  13821198    13821581    -   384 423
  his-11  II  13821778    13822314    +   537 1
  
  Why such a low assignment rate, why so little counts?
  I see that hisat2 decided to split an important proportion of the reads between orthologs, like "his-16" and "his-12", but my understanding is that the default values for the minOverlap and fracOverlap parameters should ensure that such split reads will be counted:
    --minOverlap <int>  Minimum number of overlapping bases in a read that is
                        required for read assignment. 1 by default. Number of
                        overlapping bases is counted from both reads if paired
                        end. If a negative value is provided, then a gap of up
                        to specified size will be allowed between read and the
                        feature that the read is assigned to.
  
    --fracOverlap <float> Minimum fraction of overlapping bases in a read that is
                        required for read assignment. Value should be within range
                        [0,1]. 0 by default. Number of overlapping bases is
                        counted from both reads if paired end. Both this option
                        and '--minOverlap' option need to be satisfied for read
                        assignment.
  
  What did I get wrong?
  
  user77: Given the high level of multimapping in this region, you'll need to use the -M --primary options if you want to keep many of the alignments. I would be very hesitant to use these numbers as input for DESeq2 or similar programs, since it's fairly questionable whether one should fully trust the "randomness" of the aligner's assignments. I'm more comfortable using something like salmon for such cases.
  
original_text: 292: <p>I have surprisingly low counts when running <code>featureCounts</code> on some (single-end) RNA-seq data mapped on <em>C. elegans</em> genome using <code>hisat2</code>.</p>
  
  <p>To more easily show the problem, I generated a small subset of the bam file and of the annotation file I'm using. Here is what I can see when loading these two files on IGV:</p>
  
  <p><a href="https://i.stack.imgur.com/iijez.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/iijez.png" alt="View of the his-16 -&gt; his-12 region in IGV"></a></p>
  
  <p>The coverage for the "his-11" and "his-15" genes peak at around 3600. For "his-12" and "his-16", it peaks at more than 1000</p>
  
  <p>Here are the content of my annotation file:</p>
  
  <pre><code>$ cat his_11-16.gtf 
  II  ensembl transcript  13817759    13818143    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001890"; gene_name "his-16"; gene_source "ensembl"; gene_version "1"; p_id "P8354"; transcript_biotype "protein_coding"; transcript_id "ZK131.10"; transcript_name "ZK131.10"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS11539";
  II  ensembl transcript  13818371    13818739    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001889"; gene_name "his-15"; gene_source "ensembl"; gene_version "1"; p_id "P8185"; transcript_biotype "protein_coding"; transcript_id "ZK131.9"; transcript_name "ZK131.9"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS52436";
  II  ensembl transcript  13819626    13819937    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001888"; gene_name "his-14"; gene_source "ensembl"; gene_version "1"; p_id "P21890"; transcript_biotype "protein_coding"; transcript_id "ZK131.8"; transcript_name "ZK131.8"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS50308";
  II  ensembl transcript  13820210    13820620    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001887"; gene_name "his-13"; gene_source "ensembl"; gene_version "1"; p_id "P2149"; transcript_biotype "protein_coding"; transcript_id "ZK131.7"; transcript_name "ZK131.7"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS35106";
  II  ensembl transcript  13821198    13821581    .   -   .   gene_biotype "protein_coding"; gene_id "WBGene00001886"; gene_name "his-12"; gene_source "ensembl"; gene_version "1"; p_id "P26082"; transcript_biotype "protein_coding"; transcript_id "ZK131.6"; transcript_name "ZK131.6"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS23693";
  II  ensembl transcript  13821778    13822314    .   +   .   gene_biotype "protein_coding"; gene_id "WBGene00001885"; gene_name "his-11"; gene_source "ensembl"; gene_version "1"; p_id "P10535"; transcript_biotype "protein_coding"; transcript_id "ZK131.5"; transcript_name "ZK131.5"; transcript_source "ensembl"; transcript_version "1"; tss_id "TSS52792";
  </code></pre>
  
  <p>The <code>featureCounts</code> run:</p>
  
  <pre><code>$ featureCounts -a his_11-16.gtf -o his_11-16_counts.txt -t transcript -g gene_name -O his_11-16_sorted.bam
  
          ==========     _____ _    _ ____  _____  ______          _____  
          =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
            =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
              ====      \___ \| |  | |  _ &lt;|  _  /|  __|   / /\ \ | |  | |
                ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
          ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
        v1.5.2
  
  //========================== featureCounts setting ===========================\\
  ||                                                                            ||
  ||             Input files : 1 BAM file                                       ||
  ||                           S his_11-16_sorted.bam                           ||
  ||                                                                            ||
  ||             Output file : his_11-16_counts.txt                             ||
  ||                 Summary : his_11-16_counts.txt.summary                     ||
  ||              Annotation : his_11-16.gtf (GTF)                              ||
  ||      Dir for temp files : ./                                               ||
  ||                                                                            ||
  ||                 Threads : 1                                                ||
  ||                   Level : meta-feature level                               ||
  ||              Paired-end : no                                               ||
  ||         Strand specific : no                                               ||
  ||      Multimapping reads : not counted                                      ||
  || Multi-overlapping reads : counted                                          ||
  ||   Min overlapping bases : 1                                                ||
  ||                                                                            ||
  \\===================== http://subread.sourceforge.net/ ======================//
  
  //================================= Running ==================================\\
  ||                                                                            ||
  || Load annotation file his_11-16.gtf ...                                     ||
  ||    Features : 6                                                            ||
  ||    Meta-features : 6                                                       ||
  ||    Chromosomes/contigs : 1                                                 ||
  ||                                                                            ||
  || Process BAM file his_11-16_sorted.bam...                                   ||
  ||    Single-end reads are included.                                          ||
  ||    Assign reads to features...                                             ||
  ||    Total reads : 35037                                                     ||
  ||    Successfully assigned reads : 1849 (5.3%)                               ||
  ||    Running time : 0.00 minutes                                             ||
  ||                                                                            ||
  ||                         Read assignment finished.                          ||
  ||                                                                            ||
  || Summary of counting results can be found in file "his_11-16_counts.txt"    ||
  ||                                                                            ||
  \\===================== http://subread.sourceforge.net/ ======================//
  </code></pre>
  
  <p>And the resulting counts file:</p>
  
  <pre><code>$ cat his_11-16_counts.txt
  # Program:featureCounts v1.5.2; Command:"featureCounts" "-a" "his_11-16.gtf" "-o" "his_11-16_counts.txt" "-t" "transcript" "-g" "gene_name" "-O" "his_11-16_sorted.bam" 
  Geneid  Chr Start   End Strand  Length  his_11-16_sorted.bam
  his-16  II  13817759    13818143    -   385 869
  his-15  II  13818371    13818739    +   369 3
  his-14  II  13819626    13819937    -   312 953
  his-13  II  13820210    13820620    +   411 23
  his-12  II  13821198    13821581    -   384 423
  his-11  II  13821778    13822314    +   537 1
  </code></pre>
  
  <p><strong>Why such a low assignment rate, why so little counts?</strong></p>
  
  <p>I see that <code>hisat2</code> decided to split an important proportion of the reads between orthologs, like "his-16" and "his-12", but my understanding is that the default values for the <code>minOverlap</code> and <code>fracOverlap</code> parameters should ensure that such split reads will be counted:</p>
  
  <pre><code>  --minOverlap &lt;int&gt;  Minimum number of overlapping bases in a read that is
                        required for read assignment. 1 by default. Number of
                        overlapping bases is counted from both reads if paired
                        end. If a negative value is provided, then a gap of up
                        to specified size will be allowed between read and the
                        feature that the read is assigned to.
  
    --fracOverlap &lt;float&gt; Minimum fraction of overlapping bases in a read that is
                        required for read assignment. Value should be within range
                        [0,1]. 0 by default. Number of overlapping bases is
                        counted from both reads if paired end. Both this option
                        and '--minOverlap' option need to be satisfied for read
                        assignment.
  </code></pre>
  
  <p>What did I get wrong?</p>
   77: <p>Given the high level of multimapping in this region, you'll need to use the <code>-M --primary</code> options if you want to keep many of the alignments. I would be very hesitant to use these numbers as input for DESeq2 or similar programs, since it's fairly questionable whether one should fully trust the "randomness" of the aligner's assignments. I'm more comfortable using something like <a href="http://salmon.readthedocs.io/en/latest/" rel="nofollow noreferrer">salmon</a> for such cases.</p>
  