Annotation using QSEA package
1
0
Entering edit mode
rtrivedi1 ▴ 10
@4899789e
Last seen 16 days ago
United States

Hi everyone,

I am analyzing methylation data obtained by using enrichment based methods using QSEA package. While performing annotation, I am getting the following error.

result=makeTable(QSEAset, glm=qseaGLM,
+                  groupMeans=getSampleGroups(QSEAset), keep=sig,
+                  annotation=hg38_anno_final,
+                  norm_method="beta")

Run:
adding test results from TvN
obtaining raw values for 3 samples in 1353 windows
deriving beta values...
adding annotation
Error in h(simpleError(msg, call)) : 
  error in evaluating the argument 'x' in selecting a method for function 'as.data.frame': sequences chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22 have incompatible genomes:
  - in 'x': BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38
  - in 'y': BSgenome.Hsapiens.UCSC.NA, BSgenome.Hsapiens.UCSC.NA, BSgen

For qsea object creation, I have used 'BSgenome.Hsapiens.UCSC.hg38', and got the annotation file (hg38_anno_final) using 'TxDb.Hsapiens.UCSC.hg38.knownGene'. I also have changed all the seqinfo in the hg38_anno_final file using following

seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome<-paste0("BSgenome.Hsapiens.UCSC.",seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome)

I am unable to figure out the origin of problem. Any help will be highly appreciated.

sessionInfo( )

R version 4.0.5 (2021-03-31)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Catalina 10.15.7

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.0/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] stats4    parallel  stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] shiny_1.6.0                             
 [2] AnnotationHub_2.22.1                    
 [3] BiocFileCache_1.14.0                    
 [4] dbplyr_2.1.1                            
 [5] BSgenome.Hsapiens.UCSC.hg19_1.4.3       
 [6] org.Hs.eg.db_3.12.0                     
 [7] TxDb.Hsapiens.UCSC.hg38.knownGene_3.10.0
 [8] GenomicFeatures_1.42.3                  
 [9] AnnotationDbi_1.52.0                    
[10] Biobase_2.50.0                          
[11] annotatr_1.16.0                         
[12] BSgenome.Hsapiens.UCSC.hg38_1.4.3       
[13] BSgenome_1.58.0                         
[14] rtracklayer_1.50.0                      
[15] Biostrings_2.58.0                       
[16] XVector_0.30.0                          
[17] GenomicRanges_1.42.0                    
[18] GenomeInfoDb_1.26.7                     
[19] IRanges_2.24.1                          
[20] S4Vectors_0.28.1                        
[21] BiocGenerics_0.36.1                     
[22] qsea_1.16.0                             

loaded via a namespace (and not attached):
 [1] bitops_1.0-7                           
 [2] matrixStats_0.59.0                     
 [3] bit64_4.0.5                            
 [4] progress_1.2.2                         
 [5] httr_1.4.2                             
 [6] bslib_0.2.5.1                          
 [7] tools_4.0.5                            
 [8] DT_0.18                                
 [9] utf8_1.2.1                             
[10] R6_2.5.0                               
[11] colorspace_2.0-1                       
[12] DBI_1.1.1                              
[13] withr_2.4.2                            
[14] tidyselect_1.1.1                       
[15] prettyunits_1.1.1                      
[16] bit_4.0.4                              
[17] curl_4.3.1                             
[18] compiler_4.0.5                         
[19] cli_2.5.0                              
[20] xml2_1.3.2                             
[21] DelayedArray_0.16.3                    
[22] sass_0.4.0                             
[23] scales_1.1.1                           
[24] readr_1.4.0                            
[25] askpass_1.1                            
[26] rappdirs_0.3.3                         
[27] stringr_1.4.0                          
[28] digest_0.6.27                          
[29] Rsamtools_2.6.0                        
[30] pkgconfig_2.0.3                        
[31] htmltools_0.5.1.1                      
[32] MatrixGenerics_1.2.1                   
[33] regioneR_1.22.0                        
[34] fastmap_1.1.0                          
[35] limma_3.46.0                           
[36] htmlwidgets_1.5.3                      
[37] rlang_0.4.11                           
[38] rstudioapi_0.13                        
[39] RSQLite_2.2.7                          
[40] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2
[41] jquerylib_0.1.4                        
[42] generics_0.1.0                         
[43] jsonlite_1.7.2                         
[44] zoo_1.8-9                              
[45] crosstalk_1.1.1                        
[46] BiocParallel_1.24.1                    
[47] gtools_3.9.2                           
[48] dplyr_1.0.6                            
[49] RCurl_1.98-1.3                         
[50] magrittr_2.0.1                         
[51] GenomeInfoDbData_1.2.4                 
[52] Matrix_1.3-4                           
[53] Rcpp_1.0.6                             
[54] munsell_0.5.0                          
[55] fansi_0.5.0                            
[56] lifecycle_1.0.0                        
[57] stringi_1.6.2                          
[58] yaml_2.2.1                             
[59] SummarizedExperiment_1.20.0            
[60] zlibbioc_1.36.0                        
[61] plyr_1.8.6                             
[62] HMMcopy_1.32.0                         
[63] grid_4.0.5                             
[64] blob_1.2.1                             
[65] promises_1.2.0.1                       
[66] crayon_1.4.1                           
[67] lattice_0.20-44                        
[68] hms_1.1.0                              
[69] knitr_1.33                             
[70] pillar_1.6.1                           
[71] reshape2_1.4.4                         
[72] biomaRt_2.46.3                         
[73] XML_3.99-0.6                           
[74] glue_1.4.2                             
[75] BiocVersion_3.12.0                     
[76] data.table_1.14.0                      
[77] BiocManager_1.30.16                    
[78] vctrs_0.3.8                            
[79] httpuv_1.6.1                           
[80] gtable_0.3.0                           
[81] openssl_1.4.4                          
[82] purrr_0.3.4                            
[83] assertthat_0.2.1                       
[84] cachem_1.0.5                           
[85] ggplot2_3.3.4                          
[86] xfun_0.24                              
[87] mime_0.10                              
[88] xtable_1.8-4                           
[89] later_1.2.0                            
[90] tibble_3.1.2                           
[91] GenomicAlignments_1.26.0               
[92] memoise_2.0.0                          
[93] ellipsis_0.3.2                         
[94] interactiveDisplayBase_1.28.0

Thanks

Rakesh

DNAMethylation QSEA MEDIP • 224 views
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1
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What is class(hg38_anno_final) ? It should be a GRanges object to work correctly

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It's a list of GRanges object.

> class(hg38_anno_final)
[1] "list"

> head(hg38_anno_final)
$hg38_genes_1to5kb
GRanges object with 219477 ranges and 5 metadata columns:
           seqnames            ranges strand |            id             tx_id
              <Rle>         <IRanges>  <Rle> |   <character>       <character>
       [1]     chr1        6869-10868      + |      1to5kb:1 ENST00000456328.2
       [2]     chr1        7010-11009      + |      1to5kb:2 ENST00000450305.2
       [3]     chr1       24554-28553      + |      1to5kb:3 ENST00000473358.1
       [4]     chr1       25267-29266      + |      1to5kb:4 ENST00000469289.1
       [5]     chr1       25366-29365      + |      1to5kb:5 ENST00000607096.1
       ...      ...               ...    ... .           ...               ...
  [219473]    chr22 50784287-50788286      - | 1to5kb:219473 ENST00000464678.5
  [219474]    chr22 50784631-50788630      - | 1to5kb:219474 ENST00000395590.5
  [219475]    chr22 50784601-50788600      - | 1to5kb:219475 ENST00000468451.1
  [219476]    chr22 50784631-50788630      - | 1to5kb:219476 ENST00000464740.1
  [219477]    chr22 50784046-50788045      - | 1to5kb:219477 ENST00000413505.1
               gene_id      symbol              type
           <character> <character>       <character>
       [1]   100287102     DDX11L1 hg38_genes_1to5kb
       [2]   100287102     DDX11L1 hg38_genes_1to5kb
       [3]        <NA>        <NA> hg38_genes_1to5kb
       [4]        <NA>        <NA> hg38_genes_1to5kb
       [5]   100302278   MIR1302-2 hg38_genes_1to5kb
       ...         ...         ...               ...
  [219473]       11158      RABL2B hg38_genes_1to5kb
  [219474]       11158      RABL2B hg38_genes_1to5kb
  [219475]       11158      RABL2B hg38_genes_1to5kb
  [219476]       11158      RABL2B hg38_genes_1to5kb
  [219477]       11158      RABL2B hg38_genes_1to5kb
  -------
  seqinfo: 22 sequences from BSgenome.Hsapiens.UCSC.hg38 genome

Thanks

Rakesh

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0
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What is the code you used to construct your hg38_anno_final ?

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I used the following code to get hg38_anno_final:

library(annotatr)

library(TxDb.Hsapiens.UCSC.hg38.knownGene)

hg38_annot <- builtin_annotations()[grep("hg38",builtin_annotations())]

hg38_annot <- hg38_annot[hg38_annot!="hg38_lncrna_gencode"]

an_fun <- function(anno) build_annotations(genome='hg38', annotations = anno)

hg38_anno_final <- sapply(hg38_annot,an_fun)
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1
Entering edit mode
@james-w-macdonald-5106
Last seen 32 minutes ago
United States

This sort of thing:

seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome<-paste0("BSgenome.Hsapiens.UCSC.",seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome)

is something that you should almost never (like 99% of the time) need to do, nor should you do it. In other words, mucking about in an S4 object instead of using the provided accessors is a suboptimal thing to do, unless you are a super l33t expert. Because what you ended up doing was borking all of the seqinfo for your hg38_anno_final object. Which is what the error says:

- in 'y': BSgenome.Hsapiens.UCSC.NA, BSgenome.Hsapiens.UCSC.NA, BSgen

You should probably show why you are trying to change the seqinfo slot like that, and perhaps people can then tell you the right way to do it.

0
Entering edit mode

Hi,

Step 1: I used the following code to get hg38_anno_final:

library(annotatr)

library(TxDb.Hsapiens.UCSC.hg38.knownGene)

hg38_annot <- builtin_annotations()[grep("hg38",builtin_annotations())]

hg38_annot <- hg38_annot[hg38_annot!="hg38_lncrna_gencode"]

an_fun <- function(anno) build_annotations(genome='hg38', annotations = anno)

hg38_anno_final <- sapply(hg38_annot,an_fun)

Step 2 : I calculated regions of interest (ROIs) by using this code:

CGIprom = intersect(hg38_anno_final$hg38_cpg_islands, hg38_anno_final$hg38_genes_1to5kb,ignore.strand=TRUE)

Step 3: I calculated PCA using this:

pca_cgi = getPCA(QSEAset, norm_method="beta", ROIs=CGIprom)

Error I got:

Error in mergeNamedAtomicVectors(genome(x), genome(y), what = c("sequence",  : 
  sequences chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22 have incompatible genomes:
  - in 'x': BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38
  - in 'y': hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38, hg38

So I changed the all the seqinfo slot using similar type of code:

seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome<-paste0("BSgenome.Hsapiens.UCSC.",seqinfo(hg38_anno_final$hg38_genes_5UTRs)@genome)

Then Step 3 ran successfully. without any errors.

Step 4: I performed Differential Methylation Analysis using following code:

design=model.matrix(~group, getSampleTable(QSEAset) )

qseaGLM=fitNBglm(QSEAset, design, norm_method="beta")

qseaGLM=addContrast(QSEAset, qseaGLM, coef=2, name="TvN" )

Step 5 : I started annotating the results:

library(GenomicRanges) 

sig=isSignificant(qseaGLM, fdr_th=.01)             # Step executed successfully

result=makeTable(prad_QSEAset, glm=prad_qseaGLM,                               # Step giving Errors
                 groupMeans=getSampleGroups(prad_QSEAset), keep=sig,
                 annotation=hg38_anno_final,
                 norm_method="beta")

Here I got this error:

result=makeTable(QSEAset, glm=qseaGLM,
+                  groupMeans=getSampleGroups(QSEAset), keep=sig,
+                  annotation=hg38_anno_final,
+                  norm_method="beta")

Run:
adding test results from TvN
obtaining raw values for 3 samples in 1353 windows
deriving beta values...
adding annotation
Error in h(simpleError(msg, call)) : 
  error in evaluating the argument 'x' in selecting a method for function 'as.data.frame': sequences chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22 have incompatible genomes:
  - in 'x': BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38, BSgenome.Hsapiens.UCSC.hg38
  - in 'y': BSgenome.Hsapiens.UCSC.NA, BSgenome.Hsapiens.UCSC.NA, BSgen

That's how I got the error.

Thanks

Rakesh

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1
Entering edit mode

OK. This is the part I meant about using an accessor. There is a function called genome that does what you have tried to do. It actually does genome(seqinfo(x)) where x is a GRanges object. So as an example

> library(TxDb.Hsapiens.UCSC.hg38.knownGene)
> z <- transcriptsBy(TxDb.Hsapiens.UCSC.hg38.knownGene)
> head(genome(z))
  chr1   chr2   chr3   chr4   chr5   chr6 
"hg38" "hg38" "hg38" "hg38" "hg38" "hg38" 
> genome(z) <- "BSgenome.Hsapiens.UCSC.hg38"
> head(genome(z))
                         chr1                          chr2 
"BSgenome.Hsapiens.UCSC.hg38" "BSgenome.Hsapiens.UCSC.hg38" 
                         chr3                          chr4 
"BSgenome.Hsapiens.UCSC.hg38" "BSgenome.Hsapiens.UCSC.hg38" 
                         chr5                          chr6 
"BSgenome.Hsapiens.UCSC.hg38" "BSgenome.Hsapiens.UCSC.hg38"

So doing something like

for(i in seq(along = hg38_anno_final))  genome(hg38_anno_final[[i]]) <- "BSgenome.Hsapiens.UCSC.hg38"

Should fix the issue.

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0
Entering edit mode

Issue is fixed now !!

Thanks

Rakesh

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