featureCounts (sourceforge) not returning any counts in count table, even though summary indicates reads were assigned?
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@darya-vanichkina-6050
Last seen 7.1 years ago
Australia/Centenary Institute Universit…

I'm having a very strange error when running featureCounts on a tiny test file: it says it's generating counts in the summary.txt file, but the 7th column of the counttable file is empty:

cat TestYA.multicounts.txt| awk '{print $7}' | sort | uniq

"--fraction"
/home/me/test/TestYAAligned.sortedByCoord.out.bam
cat TestYA.multicounts.txt.summary

Status    /home/me/test/TestYAAligned.sortedByCoord.out.bam
Assigned    18
Unassigned_Ambiguity    297
Unassigned_MultiMapping    0
Unassigned_NoFeatures    517
Unassigned_Unmapped    0
Unassigned_MappingQuality    0
Unassigned_FragmentLength    0
Unassigned_Chimera    0
Unassigned_Secondary    0
Unassigned_Nonjunction    0
Unassigned_Duplicate    0

 

Where did the 18 reads go?

 

head testYA.multicounts.txt
# Program:featureCounts v1.5.0; Command:"/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts" "-p" "-M" "--fraction" "−−minOverlap" "5" "-s" "n" "-T" "-C" "-R" "-t" "exon" "-g" "gene_id" "-a" "/home/me/mouse/GRCm38_mm10/gencode.vM8.primary_assembly.annotation.gtf" "-o" "TestYA.multicounts.txt" "/home/me/test/TestYAAligned.sortedByCoord.out.bam"
Geneid    Chr    Start    End    Strand    Length    /home/me/test/TestYAAligned.sortedByCoord.out.bam
ENSMUSG00000102693.1    chr1    3073253    3074322    +    1070
ENSMUSG00000064842.1    chr1    3102016    3102125    +    110
ENSMUSG00000051951.5    chr1;chr1;chr1;chr1    3205901;3213439;3421702;3670552    3207317;3216968;3421901;3671498    -;-;-;-    6094
ENSMUSG00000102851.1    chr1    3252757    3253236    +    480

 

 

 

 

 
featurecounts software error • 5.0k views
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Entering edit mode

You're working in bash, not R right?

From your first cat | awk | etc. you get: "--fraction"

So my guess there is something wrong with that? Maybe this argument is not correctly given? Are the dashes ok?

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Yes, I'm in bash, but, unfortunately, that's not the case. If you look at the results of my head command, I'm getting exactly what I would expect:

"--fraction" from line 1:

# Program:featureCounts v1.5.0; Command:"/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts" "-p" "-M" "--fraction" "−−minOverlap" "5" "-s" "n" "-T" "-C" "-R" "-t" "exon" "-g" "gene_id" "-a" "/home/me/mouse/GRCm38_mm10/gencode.vM8.primary_assembly.annotation.gtf" "-o" "TestYA.multicounts.txt" "/home/me/test/TestYAAligned.sortedByCoord.out.bam"

 

/home/me/test/TestYAAligned.sortedByCoord.out.bam from line2:

Geneid    Chr    Start    End    Strand    Length   /home/me/test/TestYAAligned.sortedByCoord.out.bam

 

And all of the rest of the lines of the file (wc -l identifies 47071 lines in the file, with each line looking like line 3) have nothing (not even a zero!) in the 7th field. I would think that if all was well, featureCounts would at least write all zeros ... But I can't see a message in the error file that would explain to me why it's all failing!

/Figured out below/

 

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@darya-vanichkina-6050
Last seen 7.1 years ago
Australia/Centenary Institute Universit…

OK, so I figured it out, and it was not very intuitive to debug! The problem I had was that when I copied the --minOverlap flag from the user manual pdf, it must have had some non-unicode characters which made featureCounts to think that --minOverlap and 5 (the value I set) were files, not a flag and it's value. 

Running the code below is bad:

/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts -p -M −−minOverlap 1 --fraction -s 0 -T 1 -C -R -t exon -g gene_id -a /home/me/project/me/Reference/mouse/GRCm38_mm10/gencode.vM8.primary_assembly.annotation.gtf -o TestYA.multicounts.txt /home/me/test/star/TestYAAligned.sortedByCoord.out.bam

Looking at the pre-run log you can see:

 

        ==========     _____ _    _ ____  _____  ______          _____
        =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \
          =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
            ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
              ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
        ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
      v1.5.0

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file  2 unknown files                      ||
||                           ? −−minOverlap                               || <- LOOK HERE
||                           ? 1                                              || <- HERE
||                           P /home/me/test/TestYAAligned. ... ||
||                                                                            ||
||             Output file : TestYA.multicounts.txt                           ||
||             Annotations : /home/me/project/me/Reference/m ... ||
||      Assignment details : <input_file>.featureCounts                       ||
||                                                                            ||
||                 Threads : 1                                                ||
||                   Level : meta-feature level                               ||
||              Paired-end : yes                                              ||
||         Strand specific : no                                               ||
||      Multimapping reads : counted (as fractions)                           ||
|| Multi-overlapping reads : not counted                                      ||
||       Read orientations : fr                                               ||
||                                                                            ||
||          Chimeric reads : not counted                                      ||
||        Both ends mapped : not required                                     ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

//================================= Running ==================================\\
||                                                                            ||
|| Load annotation file /home/me/project/me/Reference/mouse/ ... ||
||    Features : 701120                                                       ||
||    Meta-features : 47069                                                   ||
||    Chromosomes/contigs : 45                                                ||
||                                                                            ||
|| Process Unknown file −−minOverlap...                                   ||
||    Single-end reads are included.                                          ||
|| Failed to open file −−minOverlap                                       ||
|| No counts were generated for this file.                                    ||
||                                                                            ||
|| Process Unknown file 1...                                                  ||
||    Single-end reads are included.                                          ||
|| Failed to open file 1                                                      ||
|| No counts were generated for this file.                                    ||
||                                                                            ||
|| Process BAM file /home/me/test/star/TestYAAligned.sortedByCoo ... ||
||    Paired-end reads are included.                                          ||
||    Assign fragments (read pairs) to features...                            ||
||    Total fragments : 832                                                   ||
||    Successfully assigned fragments : 18 (2.2%)                             ||
||    Running time : 0.00 minutes                                             ||
||                                                                            ||
||                         Read assignment finished.                          ||
||                                                                            ||
\\===================== http://subread.sourceforge.net/ ======================//

When the code is run this way, no reads are reported in the output file (although in theory they should, if you look at the log!/

If, however, I paste the flag from the command line (i.e. by pasting from running featureCounts without arguments) all is well and I get

/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts --minOverlap 1 -p -M --fraction -s 0 -T 1 -C -R -t exon -g gene_id -a /home/me/project/me/Reference/mouse/GRCm38_mm10/gencode.vM8.primary_assembly.annotation.gtf -o TestYA.multicounts.txt /home/me/test/star/TestYAAligned.sortedByCoord.out.bam

 

This works! 

The relevant bit of the log file looks like this:

//========================== featureCounts setting ===========================\\
||                                                                            ||
||             Input files : 1 BAM file                                       ||
||                           P /home/me/test/TestYAAligned. ... ||
||                                                                            ||
||             Output file : TestYA.multicounts.txt                           ||
||             Annotations : /home/me/project/me/Reference/m ... ||
||      Assignment details : <input_file>.featureCounts                       ||

 

And the output of my about head/awk pipe is:

cat TestYA.multicounts.txt| awk '{print $7}' | sort | uniq
0
1
2.50
/home/me/test/TestYAAligned.sortedByCoord.out.bam
"-p"

As I would expect. 

 

Another thing I found out in the process is that if you include an incorrect flag for stranding:

/home/me/bin/subread-1.5.0-Linux-x86_64/bin/featureCounts --minOverlap 5 -p -M --fraction -s LALA -T 1 -C -R -t exon -g gene_id -a /home/me/project/me/Reference/mouse/GRCm38_mm10/gencode.vM8.primary_assembly.annotation.gtf -o TestYA.multicounts.txt /home/me/test/TestYAAligned.sortedByCoord.out.bam

Instead of flipping, featureCounts will just count the data as unstranded. An important GOTCHA ... Not sure which other variables are not tested for integrity...

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Entering edit mode

Yeah you use bash, I was asking this because this website is for R bioconductor questions ;-)

If you copy dashes from pdf files you get problems in bash, that's what I wanted to tell you. I suspected "--fraction" to cause it but it was "--minOverlap". Why some dashes are copied right and others not? I dont know!

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