Hi,

I am confused about the annotation of downstream region and it's priority in ChIPseeker. By default downstream defined from TTS to +3kb, and in my understanding, peaks overlapped with this region will be annotated as downstream if the priority of downstream is set to the first. However, nothing changed when I do that. There is my example:

Firstly, I annotated data using default priority(c("Promoter", "5UTR", "3UTR", "Exon", "Intron", "Downstream", "Intergenic"))

peakAnnolist <- lapply(samplefiles, annotatePeak, TxDb = txdb, tssRegion = c(-1000, 1000), annoDb = "org.Mm.eg.db")
# part results 
GRanges object with 2 ranges and 12 metadata columns:
      seqnames            ranges strand |                                     annotation   geneChr geneStart   geneEnd geneLength geneStrand      geneId
         <Rle>         <IRanges>  <Rle> |                                    <character> <integer> <integer> <integer>  <integer>  <integer> <character>
  [1]    chr11 28692672-28694373      * | Exon (ENSMUST00000146554.1/72818, exon 3 of 3)        11  28681564  28693276      11713          1       72818
  [2]    chr11 28697582-28698686      * |                              Distal Intergenic        11  28681564  28693276      11713          1       72818
              transcriptId distanceToTSS            ENSEMBL        SYMBOL                   GENENAME
               <character>     <numeric>        <character>   <character>                <character>
  [1] ENSMUST00000146554.1         11108 ENSMUSG00000084966 2810471M01Rik RIKEN cDNA 2810471M01 gene
  [2] ENSMUST00000146554.1         16018 ENSMUSG00000084966 2810471M01Rik RIKEN cDNA 2810471M01 gene

Peak(28692672-28694373) was overlapped with the geneEnd site and annotated as Exon(consistent with the IGV visualization). But I hoped that peaks like this could be annotated as downstream. Then I set the genomicAnnotationPriority to c("Promoter","Downstream", "5UTR", "3UTR", "Exon", "Intron", "Intergenic"):

peakAnnolist <- lapply(samplefiles, annotatePeak, TxDb = txdb, tssRegion = c(-1000, 1000), annoDb = "org.Mm.eg.db", 
                       genomicAnnotationPriority = c("Promoter","Downstream", "5UTR", "3UTR", "Intron",  "Intergenic"))
# part results
GRanges object with 2 ranges and 12 metadata columns:
      seqnames            ranges strand |                                     annotation   geneChr geneStart   geneEnd geneLength geneStrand      geneId
         <Rle>         <IRanges>  <Rle> |                                    <character> <integer> <integer> <integer>  <integer>  <integer> <character>
  [1]    chr11 28692672-28694373      * | Exon (ENSMUST00000146554.1/72818, exon 3 of 3)        11  28681564  28693276      11713          1       72818
  [2]    chr11 28697582-28698686      * |                              Distal Intergenic        11  28681564  28693276      11713          1       72818
              transcriptId distanceToTSS            ENSEMBL        SYMBOL                   GENENAME
               <character>     <numeric>        <character>   <character>                <character>
  [1] ENSMUST00000146554.1         11108 ENSMUSG00000084966 2810471M01Rik RIKEN cDNA 2810471M01 gene
  [2] ENSMUST00000146554.1         16018 ENSMUSG00000084966 2810471M01Rik RIKEN cDNA 2810471M01 gene

You can see that the result was same as the previous. And not only this peak, all the annotation results did not change. I also tried to remove the Exon from genomicAnnotationPriority, however, I failed again as it was just become 'Distal Intergenic'.

What should I do to solve this problem?

Thanks!