Site upstream of transcription start site annotated as intronic

[pkothiyal]

Hi, I tried annotating a few sites with annotateWithGeneParts and I'm getting an intron annotation even though the site is upstream of transcription start site. Here's an example:

BED12 entry for the associated transcript:

chr12      3906815 3908547 ENSMUST00000172913      0       +       3906815 3908265 0       3       68,138,295,     0,790,1437,

Site for annotation (chr12:3905814):

df <- data.frame (chr  = c("chr12"),
                  start = c(3905814),
                  end = c(3905814)
                  )

gr <- makeGRangesFromDataFrame(df, keep.extra.columns=FALSE,
                                      start.field="start",
                                      end.field="end")

as.data.frame(gr)

seqnames	start	end	width	strand
chr12	3905814	3905814	1	*

Annotate:

gene.obj=readTranscriptFeatures(mm10.bed", up.flank = 1000,down.flank = 1000, remove.unusual=TRUE, unique.prom = TRUE)
annRes=annotateWithGeneParts(gr,gene.obj)
cbind(getAssociationWithTSS(annRes), as.data.frame(getMembers(annRes)))

target.row	dist.to.feature	feature.name	feature.strand prom exon  intron
       1	-1002	ENSMUST00000172913	+               0    0    1

As seen above, the site is annotated in the intron of the transcript even though it is 1002 bases upstream of the transcription start site. The promoter flank is set at 1,000 so I understand it not being mapped to promoter but should it be intergenic instead of intronic in this case?

Thanks,
Prachi

[AstrobioMike]

Heya @pkothiyal and devs,

I happened to peek at this too, and just because of the older version of R where I tested, version 1.16.0 of genomation was installed, and this didn't happen in that version (in case it helps track down whatever is going on):

library(genomation)
packageVersion("genomation") # 1.16.0
library(GenomicRanges)
packageVersion("GenomicRanges") # 1.36.1

bed <- c("chr12", 3906815, 3908547, "ENSMUST00000172913", 0, "+", 3906815, 3908265, 0, 3, "68,138,295,", "0,790,1437,")
bed_df <- t(data.frame(bed))
write.table(bed_df, "mm10.bed", sep = "\t", quote = FALSE, row.names = FALSE, col.names = FALSE)

df <- data.frame (chr  = c("chr12"),
                  start = c(3905814),
                  end = c(3905814)
                  )

gr <- makeGRangesFromDataFrame(df, keep.extra.columns = FALSE,
                                      start.field = "start",
                                      end.field = "end")

gene.obj <- readTranscriptFeatures("mm10.bed", up.flank = 1000, down.flank = 1000, remove.unusual = TRUE, unique.prom = TRUE)

annRes <- annotateWithGeneParts(gr, gene.obj)

cbind(getAssociationWithTSS(annRes), as.data.frame(getMembers(annRes)))

#   target.row dist.to.feature       feature.name feature.strand prom exon intron
#            1           -1002 ENSMUST00000172913              +    0    0      0

sessionInfo()
# R version 3.6.3 (2020-02-29)
# Platform: x86_64-apple-darwin15.6.0 (64-bit)
# Running under: macOS  10.16
# 
# Matrix products: default
# LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib
# 
# locale:
# [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
# 
# attached base packages:
#  [1] parallel  stats4    grid      stats     graphics  grDevices utils     datasets  methods   base     
# 
# other attached packages:
# [1] GenomicRanges_1.36.1 GenomeInfoDb_1.20.0  IRanges_2.18.3       S4Vectors_0.22.1     BiocGenerics_0.30.0  genomation_1.16.0   
# 
# loaded via a namespace (and not attached):
#  [1] Rcpp_1.0.8                  lattice_0.20-45             Rsamtools_2.0.3             Biostrings_2.52.0          
#  [5] assertthat_0.2.1            utf8_1.2.2                  gridBase_0.4-7              R6_2.5.1                   
#  [9] plyr_1.8.6                  ggplot2_3.3.5               pillar_1.6.5                zlibbioc_1.30.0            
# [13] rlang_1.0.0                 rstudioapi_0.13             data.table_1.14.2           Matrix_1.2-18              
# [17] BiocParallel_1.18.1         readr_2.1.1                 stringr_1.4.0               RCurl_1.98-1.5             
# [21] bit_4.0.4                   munsell_0.5.0               DelayedArray_0.10.0         compiler_3.6.3             
# [25] rtracklayer_1.44.4          pkgconfig_2.0.3             tidyselect_1.1.1            SummarizedExperiment_1.14.1
# [29] tibble_3.1.6                GenomeInfoDbData_1.2.1      matrixStats_0.61.0          XML_3.99-0.3               
# [33] fansi_1.0.2                 withr_2.4.3                 crayon_1.4.2                dplyr_1.0.7                
# [37] tzdb_0.2.0                  GenomicAlignments_1.20.1    bitops_1.0-7                gtable_0.3.0               
# [41] lifecycle_1.0.1             DBI_1.1.2                   magrittr_2.0.2              scales_1.1.1               
# [45] KernSmooth_2.23-20          cli_3.1.1                   stringi_1.7.6               impute_1.58.0              
# [49] vroom_1.5.7                 XVector_0.24.0              reshape2_1.4.4              ellipsis_0.3.2             
# [53] generics_0.1.1              vctrs_0.3.8                 tools_3.6.3                 bit64_4.0.5                
# [57] BSgenome_1.52.0             Biobase_2.44.0              glue_1.6.1                  seqPattern_1.16.0          
# [61] purrr_0.3.4                 hms_1.1.1                   plotrix_3.8-2               colorspace_2.0-2           
# [65] BiocManager_1.30.16    

[al2na]

Thank you, something must have been changed in GenomicRanges that causes this.

On Mon, Jan 31, 2022 at 5:28 PM Mike Lee ***@***.***> wrote: Heya @pkothiyal <https://github.com/pkothiyal> and devs, I happened to peek at this too, and just because of the older version of R where I tested, version 1.16.0 of genomation was installed, and this didn't happen in that version (in case it helps track down whatever is going on): library(genomation) packageVersion("genomation") # 1.16.0 library(GenomicRanges) packageVersion("GenomicRanges") # 1.36.1 bed <- c("chr12", 3906815, 3908547, "ENSMUST00000172913", 0, "+", 3906815, 3908265, 0, 3, "68,138,295,", "0,790,1437,")bed_df <- t(data.frame(bed)) write.table(bed_df, "mm10.bed", sep = "\t", quote = FALSE, row.names = FALSE, col.names = FALSE) df <- data.frame (chr = c("chr12"), start = c(3905814), end = c(3905814) ) gr <- makeGRangesFromDataFrame(df, keep.extra.columns = FALSE, start.field = "start", end.field = "end") gene.obj <- readTranscriptFeatures("mm10.bed", up.flank = 1000, down.flank = 1000, remove.unusual = TRUE, unique.prom = TRUE) annRes <- annotateWithGeneParts(gr, gene.obj) cbind(getAssociationWithTSS(annRes), as.data.frame(getMembers(annRes))) # target.row dist.to.feature feature.name feature.strand prom exon intron# 1 -1002 ENSMUST00000172913 + 0 0 0 sessionInfo()# R version 3.6.3 (2020-02-29)# Platform: x86_64-apple-darwin15.6.0 (64-bit)# Running under: macOS 10.16# # Matrix products: default# LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib# # locale:# [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8# # attached base packages:# [1] parallel stats4 grid stats graphics grDevices utils datasets methods base # # other attached packages:# [1] GenomicRanges_1.36.1 GenomeInfoDb_1.20.0 IRanges_2.18.3 S4Vectors_0.22.1 BiocGenerics_0.30.0 genomation_1.16.0 # # loaded via a namespace (and not attached):# [1] Rcpp_1.0.8 lattice_0.20-45 Rsamtools_2.0.3 Biostrings_2.52.0 # [5] assertthat_0.2.1 utf8_1.2.2 gridBase_0.4-7 R6_2.5.1 # [9] plyr_1.8.6 ggplot2_3.3.5 pillar_1.6.5 zlibbioc_1.30.0 # [13] rlang_1.0.0 rstudioapi_0.13 data.table_1.14.2 Matrix_1.2-18 # [17] BiocParallel_1.18.1 readr_2.1.1 stringr_1.4.0 RCurl_1.98-1.5 # [21] bit_4.0.4 munsell_0.5.0 DelayedArray_0.10.0 compiler_3.6.3 # [25] rtracklayer_1.44.4 pkgconfig_2.0.3 tidyselect_1.1.1 SummarizedExperiment_1.14.1# [29] tibble_3.1.6 GenomeInfoDbData_1.2.1 matrixStats_0.61.0 XML_3.99-0.3 # [33] fansi_1.0.2 withr_2.4.3 crayon_1.4.2 dplyr_1.0.7 # [37] tzdb_0.2.0 GenomicAlignments_1.20.1 bitops_1.0-7 gtable_0.3.0 # [41] lifecycle_1.0.1 DBI_1.1.2 magrittr_2.0.2 scales_1.1.1 # [45] KernSmooth_2.23-20 cli_3.1.1 stringi_1.7.6 impute_1.58.0 # [49] vroom_1.5.7 XVector_0.24.0 reshape2_1.4.4 ellipsis_0.3.2 # [53] generics_0.1.1 vctrs_0.3.8 tools_3.6.3 bit64_4.0.5 # [57] BSgenome_1.52.0 Biobase_2.44.0 glue_1.6.1 seqPattern_1.16.0 # [61] purrr_0.3.4 hms_1.1.1 plotrix_3.8-2 colorspace_2.0-2 # [65] BiocManager_1.30.16 — Reply to this email directly, view it on GitHub <#203 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AAE32EPZRTVRET5SFUDJ4YLUY22BFANCNFSM5M3QWVXQ> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

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