I have asked this question on Biostars (https://www.biostars.org/p/361800/), and have not got any response. Many thanks for any suggestions and helps.

We have six RNA-Seq data, including three wild type and three knock-out mice. I use STAR 2.5.3a (built-in Partek Flow) for the alignment and featureCounts (Rsubread_1.30.9) for the quantification. The command I run featureCoutns is below.

files <- c("Chuong753.bam", "Chuong754.bam", "Chuong755.bam",
  "Chuong756.bam", "Chuong757.bam", "Chuong758.bam")
rc <- featureCounts(files, annot.ext = "mm10ncbiRefSeqCurated.gtf",
  isGTFAnnotationFile = TRUE, GTF.featureType = "exon",
  GTF.attrType = "gene_id", minMQS = 10, strandSpecific = 0,
  nthreads = 6, verbose = TRUE)

I found that there are a lot of Unassigned_MappingQuality reads (about 26.9% of total alignments, the detail below). Should I set minMQS=3 or 0 to increase the number of Assigned reads? Many thanks.

rc$stat
                          Status Chuong753 Chuong754 Chuong755 Chuong756 Chuong757 Chuong758
1                       Assigned  28552790  19795064  26274194  26601820  21264775  21703604
2            Unassigned_Unmapped         0         0         0         0         0         0
3      Unassigned_MappingQuality  10233734   6718392  10369784   9299763   9249801  12905108
4             Unassigned_Chimera         0         0         0         0         0         0
5      Unassigned_FragmentLength         0         0         0         0         0         0
6           Unassigned_Duplicate         0         0         0         0         0         0
7        Unassigned_MultiMapping         0         0         0         0         0         0
8           Unassigned_Secondary         0         0         0         0         0         0
9         Unassigned_Nonjunction         0         0         0         0         0         0
10         Unassigned_NoFeatures   2596367   1732967   2577948   1882419   2235492   2757615
11 Unassigned_Overlapping_Length         0         0         0         0         0         0
12          Unassigned_Ambiguity    237389    166711    228953    216951    178357    218899

In addition, I am confused about the mapping quality score. The range of mapping quality score seems:

1. 0 to 255 for STAR (http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STAR.posix/doc/STARmanual.pdf)

2. 0 to 40 for Rsubread v1.32.0 (https://bioconductor.org/packages/release/bioc/vignettes/Rsubread/inst/doc/SubreadUsersGuide.pdf)

3. 0 to read length (i.e. 75 in my case) for Rsubread v1.20.1 (http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.725.140&rep=rep1&type=pdf)

4. 0 to 200 for Rsubread v1.11.10 (http://citeseerx.ist.psu.edu/viewdochelpnload?doi=10.1.1.366.8312&rep=rep1&type=pdf)

Could you hlep me? Thanks a lot.

Please post your questions regarding Rsubread (featureCounts is part of it) to this Support Site as Rsubread is a Bioconductor package.

In general I don't recommend throwing away reads with low MAQ value when you count them to genes. This is because an RNA-seq read with low MAQ is likely to be correctly mapped if it hits an annotated gene (more precisely it hits an exon of a gene). The transcriptome only accounts for ~2% of the genome, therefore the chance that a read incorrectly mapped to the genome happens to map to the transcriptome is very low.

Regarding MAQ values reported by Rsubread, we have recently changed them to the range of 0 to 40 to make it be in line with the Phred score range.