featureCount Error "No paired-end reads were detected in paired-end read library"

Question

[cook.675]

I created a combined mm39 and MHV-A59 (Viral) reference genome and aligned my paired end reads using STAR with the following input commands:

STAR 
--runMode alignReads   
--runThreadN 16   
--genomeDir /genomeDir   
--readFilesIn /FastqDir/1.fq.gz, /FastqDir/2.fq.gz      
--readFilesCommand gunzip   -c      
--outReadsUnmapped Fastx   
--outSAMtype BAM   SortedByCoordinate 

It seems that everything went fine. Here is a result from one of the alignments:

                                 Started job on |   Nov 14 20:08:30
                         Started mapping on |   Nov 14 20:09:03
                                Finished on |   Nov 14 20:15:12
   Mapping speed, Million of reads per hour |   808.26

                      Number of input reads |   82847062
                  Average input read length |   150
                                UNIQUE READS:
               Uniquely mapped reads number |   77656936
                    Uniquely mapped reads % |   93.74%
                      Average mapped length |   149.28
                   Number of splices: Total |   37645057
        Number of splices: Annotated (sjdb) |   37376062
                   Number of splices: GT/AG |   37313095
                   Number of splices: GC/AG |   265167
                   Number of splices: AT/AC |   28575
           Number of splices: Non-canonical |   38220
                  Mismatch rate per base, % |   0.26%
                     Deletion rate per base |   0.01%
                    Deletion average length |   1.82
                    Insertion rate per base |   0.01%
                   Insertion average length |   1.57
                         MULTI-MAPPING READS:
    Number of reads mapped to multiple loci |   3072281
         % of reads mapped to multiple loci |   3.71%
    Number of reads mapped to too many loci |   98073
         % of reads mapped to too many loci |   0.12%
                              UNMAPPED READS:
   % of reads unmapped: too many mismatches |   0.00%
             % of reads unmapped: too short |   2.37%
                 % of reads unmapped: other |   0.07%
                              CHIMERIC READS:
                   Number of chimeric reads |   0
                        % of chimeric reads |   0.00%

Now I take this BAM file and run it through featureCount with the following parameters, where combined gtf is my mm39 and MHV-A59 gtf file I used for making the index:

featureCounts(files="/Users/BamFile.bam",
                          annot.ext = "/Users/combined.gtf", 
                          isGTFAnnotationFile = TRUE,
                          isPairedEnd=TRUE, 
                          requireBothEndsMapped = TRUE,
                          countReadPairs = TRUE,
                          strandSpecific = 2)

And I get the following error: ERROR: No paired-end reads were detected in paired-end read library

I feel like this must be a result of the combined mouse and viral reference but I cant quite figure out what it wrong. I checked and it looks like I have reads mapped to the viral genome so I think everything went smoothly on the mapping side. Any advice would be greatly appreciated!!!

Answer 1

[rpolicastro]

The R1 and R2 reads should be space separated for paired end alignment. If you comma separate them they get concatenated and treated as one file.

[cook.675]

beautiful thank you so much!