Hello,

I am currently using the ChIPseeker package to analyze ChIP-seq data of mine. I am working with data derived from the plant of Lotus japonicus. Thus, I had to convert the corresponding .GFF3 file from Lotus to TxDb by using the makeTxDbFromGFF in the GenomicFeatures package. I also had to use MeSH.Miy.eg.db as annoDb= in annotatePeak function in order to annotate my peaks:

peakAnno <- annotatePeak(files[[1]], tssRegion=c(-3000, 3000), TxDb=txdb, annoDb="MeSH.Miy.eg.db").

I went through almost all the step of the manual and it did worked well, except the Funtional enrichment analysis, which I need it. I just paste below my script about it:

library(ChIPseeker) peakAnno <- annotatePeak(files[[3]], tssRegion=c(-3000, 3000), TxDb=txdb, annoDb="MeSH.Miy.eg.db")

loading peak file... 2020-05-08 07:37:57 preparing features information... 2020-05-08 07:37:58 identifying nearest features... 2020-05-08 07:37:58 calculating distance from peak to TSS... 2020-05-08 07:38:01 assigning genomic annotation... 2020-05-08 07:38:01 adding gene annotation... 2020-05-08 07:38:11 assigning chromosome lengths 2020-05-08 07:38:11 done... 2020-05-08 07:38:11 Warning message: In annotatePeak(files[[3]], tssRegion = c(-3000, 3000), TxDb = txdb, : Unknown ID type, gene annotation will not be added... library(ReactomePA) ReactomePA v1.24.0 For help: https://guangchuangyu.github.io/ReactomePA

If you use ReactomePA in published research, please cite: Guangchuang Yu, Qing-Yu He. ReactomePA: an R/Bioconductor package for reactome pathway analysis and visualization. Molecular BioSystems 2016, 12(2):477-479

pathway1 <- enrichPathway(as.data.frame(peakAnno)$geneId) Loading required package: org.Hs.eg.db

--> No gene can be mapped.... --> Expected input gene ID: 3699,5834,221037,2665,6240,2793 --> return NULL...

head(pathway1, 2) NULL gene <- seq2gene(peak, tssRegion = c(-1000, 1000), flankDistance = 3000, TxDb=txdb) pathway2 <- enrichPathway(gene) --> No gene can be mapped.... --> Expected input gene ID: 4723,51311,57406,6383,65977,80143 --> return NULL... head(pathway2, 2) NULL

I have two questions if you would like to answer for me:

1) I understand that ReactomePA (suggested to be used from the ChIPseeker manual) does not support my organism (Lotus japonicus). But I do not how else I can make the Functional enrichment analysis of the ChIPseeker work. Could you please help on that?

2) I do use the results of the annotatePeak function (peakAnno) for producing successfully other data (e..g Disrtibution of the peaks on different genetic elements or relative to TSS and more), but since I get the massage:

Warning message: In annotatePeak(files[[3]], tssRegion = c(-3000, 3000), TxDb = txdb, : Unknown ID type, gene annotation will not be added...

after I run the annotatePeak function, I am wondering if whatever is produced downstream of that is correct or not? Any help will be appreciable, Best, Manolis