Wrong annotation (distal intergenic instead of downstream) with annotatePeak

[beryljones]

Hello,

Thank you very much for this package! I seem to have an error in the annotatePeak output that I cannot find an existing issue about. I am using R version 4.1.2 and ChIPseeker_1.30.3. Here is the rest of the relevant session_info():

#R version 4.1.2 (2021-11-01)
#Platform: x86_64-w64-mingw32/x64 (64-bit)
#Running under: Windows 10 x64 (build 19044)

#Matrix products: default

#locale
LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252 LC_MONETARY=English_United States.1252
LC_NUMERIC=C LC_TIME=English_United States.1252

#attached base packages:
stats4 stats graphics grDevices utils datasets methods base

#other attached packages:
ggimage_0.3.1 ggplot2_3.4.0 ggupset_0.3.0 GenomicFeatures_1.46.5 AnnotationDbi_1.56.2
Biobase_2.54.0 GenomicRanges_1.46.1 GenomeInfoDb_1.30.0 IRanges_2.28.0 S4Vectors_0.32.2
BiocGenerics_0.40.0 ChIPseeker_1.30.3

I have a custom genome/annotation, so I first make a txdb object from my gff3:

library(GenomicFeatures)
test_txdb <- makeTxDbFromGFF(file="chr3_subset.gff3",
dataSource="chr3_subset.gff3",
organism="Lasioglossum albipes")

Here are a list of regions I want to annotate (small subset to illustrate the issue):
LALB_chr_3 2627151 2628080
LALB_chr_3 2659532 2660013
LALB_chr_3 2664362 2666431

And here are relevant proximal genes (just showing the "gene" entry from the gff3):
LALB_chr_3 maker gene 2623697 2633465 . - . ID=LALB_04121;Name=LALB_04121
LALB_chr_3 maker gene 2660123 2661437 . - . ID=LALB_04122;Name=LALB_04122

I would like to annotate a region as downstream if it is within 5000 bp of the end of the gene (on either strand). I found elsewhere on this site that I can use the options(ChIPseeker.downstreamDistance = 5000) to accomplish this. Here is what I run:

options(ChIPseeker.downstreamDistance = 5000)
peakAnno <- annotatePeak("test_chr3_regions.bed",
level="gene",
sameStrand=FALSE,
ignoreDownstream=FALSE,
ignoreUpstream=FALSE,
assignGenomicAnnotation=TRUE,
addFlankGeneInfo=TRUE,
overlap="all",
flankDistance = 10000,
genomicAnnotationPriority = c("Promoter", "Intron", "5UTR", "3UTR", "Exon", "Downstream", "Intergenic"),
tssRegion=c(-10000, 0),TxDb=test_txdb, verbose=TRUE)

Here is the output:
seqnames start end width strand annotation geneChr geneStart geneEnd
1 LALB_chr_3 2627152 2628080 929 * Intron (LALB_04121-RA/LALB_04121, intron 3 of 5) 1 2623697 2633465
2 LALB_chr_3 2659533 2660013 481 * Distal Intergenic 1 2660123 2661437
3 LALB_chr_3 2664363 2666431 2069 * Promoter (2-3kb) 1 2660123 2661437
geneLength geneStrand geneId distanceToTSS flank_geneIds flank_gene_distances
1 9769 2 LALB_04121 5385 LALB_04121 0
2 1315 2 LALB_04122 1424 LALB_04122 1424
3 1315 2 LALB_04122 -2926 LALB_04122 -2926

The second region is annotated as "Distal Intergenic", but it is only 110bp downstream from the gene LALB_04122. Even the default downstream distance (300, I think) should be greater than 110bp, so the options(ChIPseeker.downstreamDistance = 5000) should not matter here, although I do want to include regions that are within 5kb.

Happy to send files if needed or answer any other questions. Thank you!