gene annotation for non-model species #1352

pengxin2019 · 2022-03-22T21:20:11Z

pengxin2019
Mar 22, 2022

Hi Ryan,
I am working on pig scATAC data.

the packages i used to generate the annotation is like this:

library(TxDb.Sscrofa.UCSC.susScr11.refGene)
library(org.Ss.eg.db)
library(BSgenome.Sscrofa.UCSC.susScr11)

genomeAnnotation <- createGenomeAnnotation(genome = BSgenome.Sscrofa.UCSC.susScr11)
geneAnnotation <- createGeneAnnotation(TxDb = TxDb.Sscrofa.UCSC.susScr11.refGene, OrgDb = org.Ss.eg.db)

However, there are only 4364 genes found which worries me a lot since it much less than the number i expected (I downloaded gtf file and use it as annotation when I used Signac and there are around 15k genes). As a result, when I ran addGeneIntegrationMatrix (the inputs are an ArchRProj object and an Seurat object. This Seurat scRNA object has 20k genes), only around 3k overlapping genes are found.

getGenes(proj.doublet.filtered.harmony)
# GRanges object with 4364 ranges and 2 metadata columns:
#   seqnames              ranges strand |     gene_id      symbol
# <Rle>           <IRanges>  <Rle> | <character> <character>
#   [1]     chr1         23827-40033      + |   100621969       PSMB1
# [2]     chr1     1013707-1013802      - |   104796093     MIR9815
# [3]     chr1     1340183-1340263      - |   104797359     MIR9821
# [4]     chr1     1665568-1665643      - |   104796099     MIR9824
# [5]     chr1     2541537-2552860      + |   100513634        MPC1
# ...      ...                 ...    ... .         ...         ...
# [4360]     chrX 125546698-125560059      - |   100516055       CLIC2
# [4361]     chrX 125863303-125917224      + |   100627083       VAMP7
# [4362]     chrY     9687854-9704413      - |   100625415      EIF1AY
# [4363]     chrY   10124811-10132684      + |      396714       AMELY
# [4364]     chrY   40484990-40485700      - |      407740         SRY
# -------
#   seqinfo: 20 sequences from susScr11 genome


proj.doublet.filtered.harmony <- addGeneIntegrationMatrix(
  ArchRProj = proj.doublet.filtered.harmony, 
  useMatrix = "GeneScoreMatrix",
  matrixName = "GeneIntegrationMatrix",
  reducedDims = "Harmony",
  seRNA = pbmc_sc_rna,
  addToArrow = FALSE,
  groupRNA = "shorten_cell_types",#A column name in metadata of SeuratObject) of seRNA that will be used to determine the subgroupings specified in groupList
  nameCell = "predictedCell_Un",#A column name to add to cellColData for the predicted scRNA-seq cell in the specified ArchRProject. This is useful for identifying which cell was closest to the scATAC-seq cell.
  nameGroup = "predictedGroup_Un",
  nameScore = "predictedScore_Un"
)
# 2022-03-22 15:50:27 : Found 3047 overlapping gene names from gene scores and rna matrix!, 0.061 mins elapsed.

Is that possible to use gtf file to do the annotation which might bring me much more genes?

Thanks

rcorces · 2022-03-22T23:36:11Z

rcorces
Mar 22, 2022
Maintainer

Just to clarify for any other users who come across this, ArchR doesnt manage gene annotations. So the reason there are only ~4000 genes is because TxDb.Sscrofa.UCSC.susScr11.refGene only has that many genes.

You can create your own custom gene annotation if you have gene coordinates, exon coordinates, and the TSS coordinates. This is outlined in section 1.5.1 of the manual
https://www.archrproject.com/bookdown/getting-set-up.html

geneAnnotation <- createGeneAnnotation(
  TSS = geneAnnotation$TSS, 
  exons = geneAnnotation$exons, 
  genes = geneAnnotation$genes
)

4 replies

pengxin2019 Mar 23, 2022
Author

Hi Ryan,
Thanks for quick response again.
I tried the script below.
Below are my genome and gene annotation information.

# > BSgenome.Sscrofa.UCSC.susScr11
# Pig genome:
#   # organism: Sus scrofa (Pig)
#   # genome: susScr11
#   # provider: UCSC
#   # release date: Feb. 2017
#   # 613 sequences:
#   #   chr1                       chr2                      
#   #   chr3                       chr4                      
#   #   chr5                       chr6                      
#   #   chr7                       chr8                      
#   #   chr9                       chr10                     
#   #   ...                        ...                       
# #   chrUn_NW_018085360v1       chrUn_NW_018085361v1  

genomeAnnotation <- createGenomeAnnotation(genome = BSgenome.Sscrofa.UCSC.susScr11)
gtf.gr <- makeGRangesFromDataFrame(gtf.df, keep.extra.columns=TRUE)
> gtf.gr
GRanges object with 1530978 ranges and 21 metadata columns:
                     seqnames    ranges strand |   source       type     score
                        <Rle> <IRanges>  <Rle> | <factor>   <factor> <numeric>
        [1]              chr1    1-3782      + |  ensembl gene              NA
        [2]              chr1    1-3780      + |  ensembl transcript        NA
        [3]              chr1    1-2465      + |  ensembl exon              NA
        [4]              chr1 3118-3780      + |  ensembl exon              NA
        [5]              chr1  792-3782      + |  ensembl transcript        NA
        ...               ...       ...    ... .      ...        ...       ...
  [1530974] chrAEMK02000302.1 5535-5587      - |  ensembl exon              NA
  [1530975] chrAEMK02000302.1      4-47      - |  ensembl exon              NA
  [1530976] chrAEMK02000302.1    4-8300      - |  ensembl transcript        NA
  [1530977] chrAEMK02000302.1 8159-8300      - |  ensembl exon              NA
  [1530978] chrAEMK02000302.1      4-47      - |  ensembl exon              NA
                phase            gene_id gene_version gene_source gene_biotype
            <integer>        <character>  <character> <character>  <character>
        [1]      <NA> ENSSSCG00000048769            1     ensembl       lncRNA
        [2]      <NA> ENSSSCG00000048769            1     ensembl       lncRNA
        [3]      <NA> ENSSSCG00000048769            1     ensembl       lncRNA
        [4]      <NA> ENSSSCG00000048769            1     ensembl       lncRNA
        [5]      <NA> ENSSSCG00000048769            1     ensembl       lncRNA
        ...       ...                ...          ...         ...          ...
  [1530974]      <NA> ENSSSCG00000049924            1     ensembl       lncRNA
  [1530975]      <NA> ENSSSCG00000049924            1     ensembl       lncRNA
  [1530976]      <NA> ENSSSCG00000049924            1     ensembl       lncRNA
  [1530977]      <NA> ENSSSCG00000049924            1     ensembl       lncRNA
  [1530978]      <NA> ENSSSCG00000049924            1     ensembl       lncRNA
                 transcript_id transcript_version transcript_source
                   <character>        <character>       <character>
        [1]               <NA>               <NA>              <NA>
        [2] ENSSSCT00000066540                  1           ensembl
        [3] ENSSSCT00000066540                  1           ensembl
        [4] ENSSSCT00000066540                  1           ensembl
        [5] ENSSSCT00000078085                  1           ensembl
        ...                ...                ...               ...
  [1530974] ENSSSCT00000066525                  1           ensembl
  [1530975] ENSSSCT00000066525                  1           ensembl
  [1530976] ENSSSCT00000068345                  1           ensembl
  [1530977] ENSSSCT00000068345                  1           ensembl
  [1530978] ENSSSCT00000068345                  1           ensembl
            transcript_biotype exon_number            exon_id exon_version
                   <character> <character>        <character>  <character>
        [1]               <NA>        <NA>               <NA>         <NA>
        [2]             lncRNA        <NA>               <NA>         <NA>
        [3]             lncRNA           1 ENSSSCE00000399651            1
        [4]             lncRNA           2 ENSSSCE00000450299            1
        [5]             lncRNA        <NA>               <NA>         <NA>
        ...                ...         ...                ...          ...
  [1530974]             lncRNA           2 ENSSSCE00000399377            1
  [1530975]             lncRNA           3 ENSSSCE00000418429            1
  [1530976]             lncRNA        <NA>               <NA>         <NA>
  [1530977]             lncRNA           1 ENSSSCE00000453271            1
  [1530978]             lncRNA           2 ENSSSCE00000418429            1
             protein_id protein_version   gene_name transcript_name
            <character>     <character> <character>     <character>
        [1]        <NA>            <NA>        <NA>            <NA>
        [2]        <NA>            <NA>        <NA>            <NA>
        [3]        <NA>            <NA>        <NA>            <NA>
        [4]        <NA>            <NA>        <NA>            <NA>
        [5]        <NA>            <NA>        <NA>            <NA>
        ...         ...             ...         ...             ...
  [1530974]        <NA>            <NA>        <NA>            <NA>
  [1530975]        <NA>            <NA>        <NA>            <NA>
  [1530976]        <NA>            <NA>        <NA>            <NA>
  [1530977]        <NA>            <NA>        <NA>            <NA>
  [1530978]        <NA>            <NA>        <NA>            <NA>
            projection_parent_transcript         tag
                             <character> <character>
        [1]                         <NA>        <NA>
        [2]                         <NA>        <NA>
        [3]                         <NA>        <NA>
        [4]                         <NA>        <NA>
        [5]                         <NA>        <NA>
        ...                          ...         ...
  [1530974]                         <NA>        <NA>
  [1530975]                         <NA>        <NA>
  [1530976]                         <NA>        <NA>
  [1530977]                         <NA>        <NA>
  [1530978]                         <NA>        <NA>
  -------
  seqinfo: 351 sequences from an unspecified genome; no seqlengths

geneAnnotation <- createGeneAnnotation(
  TSS = gtf.gr, 
  exons = gtf.gr, 
  genes = gtf.gr)

# > geneAnnotation
# List of length 3
# names(3): genes exons TSS
# #geneAnnotation

# > geneAnnotation[[3]]
>  geneAnnotation[[3]]
GRanges object with 533916 ranges and 21 metadata columns:
                    seqnames      ranges strand |   source           type
                       <Rle>   <IRanges>  <Rle> | <factor>       <factor>
       [1]              chr1      1-3782      + |  ensembl     gene      
       [2]              chr1      1-3780      + |  ensembl     transcript
       [3]              chr1      1-2465      + |  ensembl     exon      
       [4]              chr1   3118-3780      + |  ensembl     exon      
       [5]              chr1    792-3782      + |  ensembl     transcript
       ...               ...         ...    ... .      ...            ...
  [533912] chrAEMK02000695.1 14777-14844      - |  ensembl five_prime_utr
  [533913] chrAEMK02000302.1      4-8300      - |  ensembl gene          
  [533914] chrAEMK02000302.1   8159-8300      - |  ensembl exon          
  [533915] chrAEMK02000302.1   5535-5587      - |  ensembl exon          
  [533916] chrAEMK02000302.1        4-47      - |  ensembl exon          
               score     phase            gene_id gene_version gene_source
           <numeric> <integer>        <character>  <character> <character>
       [1]        NA      <NA> ENSSSCG00000048769            1     ensembl
       [2]        NA      <NA> ENSSSCG00000048769            1     ensembl
       [3]        NA      <NA> ENSSSCG00000048769            1     ensembl
       [4]        NA      <NA> ENSSSCG00000048769            1     ensembl
       [5]        NA      <NA> ENSSSCG00000048769            1     ensembl
       ...       ...       ...                ...          ...         ...
  [533912]        NA      <NA> ENSSSCG00000034846            1     ensembl
  [533913]        NA      <NA> ENSSSCG00000049924            1     ensembl
  [533914]        NA      <NA> ENSSSCG00000049924            1     ensembl
  [533915]        NA      <NA> ENSSSCG00000049924            1     ensembl
  [533916]        NA      <NA> ENSSSCG00000049924            1     ensembl
             gene_biotype      transcript_id transcript_version
              <character>        <character>        <character>
       [1]         lncRNA               <NA>               <NA>
       [2]         lncRNA ENSSSCT00000066540                  1
       [3]         lncRNA ENSSSCT00000066540                  1
       [4]         lncRNA ENSSSCT00000066540                  1
       [5]         lncRNA ENSSSCT00000078085                  1
       ...            ...                ...                ...
  [533912] protein_coding ENSSSCT00000039225                  1
  [533913]         lncRNA               <NA>               <NA>
  [533914]         lncRNA ENSSSCT00000066525                  1
  [533915]         lncRNA ENSSSCT00000066525                  1
  [533916]         lncRNA ENSSSCT00000066525                  1
           transcript_source transcript_biotype exon_number            exon_id
                 <character>        <character> <character>        <character>
       [1]              <NA>               <NA>        <NA>               <NA>
       [2]           ensembl             lncRNA        <NA>               <NA>
       [3]           ensembl             lncRNA           1 ENSSSCE00000399651
       [4]           ensembl             lncRNA           2 ENSSSCE00000450299
       [5]           ensembl             lncRNA        <NA>               <NA>
       ...               ...                ...         ...                ...
  [533912]           ensembl     protein_coding        <NA>               <NA>
  [533913]              <NA>               <NA>        <NA>               <NA>
  [533914]           ensembl             lncRNA           1 ENSSSCE00000453271
  [533915]           ensembl             lncRNA           2 ENSSSCE00000399377
  [533916]           ensembl             lncRNA           3 ENSSSCE00000418429
           exon_version  protein_id protein_version   gene_name transcript_name
            <character> <character>     <character> <character>     <character>
       [1]         <NA>        <NA>            <NA>        <NA>            <NA>
       [2]         <NA>        <NA>            <NA>        <NA>            <NA>
       [3]            1        <NA>            <NA>        <NA>            <NA>
       [4]            1        <NA>            <NA>        <NA>            <NA>
       [5]         <NA>        <NA>            <NA>        <NA>            <NA>
       ...          ...         ...             ...         ...             ...
  [533912]         <NA>        <NA>            <NA>        <NA>            <NA>
  [533913]         <NA>        <NA>            <NA>        <NA>            <NA>
  [533914]            1        <NA>            <NA>        <NA>            <NA>
  [533915]            1        <NA>            <NA>        <NA>            <NA>
  [533916]            1        <NA>            <NA>        <NA>            <NA>
           projection_parent_transcript         tag
                            <character> <character>
       [1]                         <NA>        <NA>
       [2]                         <NA>        <NA>
       [3]                         <NA>        <NA>
       [4]                         <NA>        <NA>
       [5]                         <NA>        <NA>
       ...                          ...         ...
  [533912]                         <NA>        <NA>
  [533913]                         <NA>        <NA>
  [533914]                         <NA>        <NA>
  [533915]                         <NA>        <NA>
  [533916]                         <NA>        <NA>
  -------
  seqinfo: 351 sequences from an unspecified genome; no seqlengths

geneAnnotation and genomeAnnotation so far look good.

ArrowFiles <- createArrowFiles(
  inputFiles = inputFiles,
  sampleNames = names(inputFiles),
  minTSS = 4, #Dont set this too high because you can always increase later
  minFrags = 1000, 
  addTileMat = TRUE,
  genomeAnnotation = genomeAnnotation,
  geneAnnotation = geneAnnotation
)

However, I saw those words when I created the ArrowFiles:

Found Gene Seqnames not in GenomeAnnotation chromSizes, Removing : chrMT,chrAEMK02000452.1,chrAEMK02000698.1,chrAEMK02000361.1,chrAEMK02000598.1,chrAEMK02000682.1,chrAEMK02000449.1,chrAEMK02000677.1,chrAEMK02000408.1,chrAEMK02000569.1,chrAEMK02000393.1,chrAEMK02000510.1,chrAEMK02000328.1,chrAEMK02000465.1,chrAEMK02000133.1,chrAEMK02000661.1,chrAEMK02000261.1,chrAEMK02000324.1,chrAEMK02000258.1,chrAEMK02000537.1,chrAEMK02000522.1,chrAEMK02000694.1,chrAEMK02000451.1,chrAEMK02000478.1,chrAEMK02000496.1,chrAEMK02000390.1,chrAEMK02000175.1,chrAEMK02000602.1,chrAEMK02000370.1,chrAEMK02000213.1,chrAEMK02000630.1,chrAEMK02000264.1,chrAEMK02000600.1,chrAEMK02000410.1,chrAEMK02000525.1,chrAEMK02000159.1,chrAEMK02000423.1,chrAEMK02000566.1,chrAEMK020002.. (this part makes sense to me and I am not surprised)

at the bottom there are some errors which i do not understand and do not know how to fix it:

Can you give me some further suggestions?

Thanks
Best

rcorces Mar 23, 2022
Maintainer

In the future, please try not to post screenshots of text. These are unsearchable for future users. Instead, copy and paste the text into code blocks with markdown syntax (three backticks).

Previously I pointed you to a section of the manual. Please read that section carefully. Especially 1.5.2 which talks about addArchRChrPrefix(). I believe this is your problem

pengxin2019 Mar 23, 2022
Author

Hi ,
Thanks for pointing that out. The genomeAnnotation which is BSgenome.Sscrofa.UCSC.susScr11 does have "chr" as you can see from the output above. Originally geneAnnotation which is gtf does not have prefix "chr". I added "chr" to the seqnames of gtf to make it match with genomeAnnotation.

I DID try to set up either addArchRChrPrefix(chrPrefix = FALSE) or addArchRChrPrefix(chrPrefix = TRUE) before I run createArrowFiles. But they brought me the same error:

************************************************************
2022-03-23 18:10:28 : ERROR Found in .addGeneScoreMat for (pbmc.6800.1x : 4 of 4) 
LogFile = ArchRLogs/ArchR-createArrows-7ace23bf42f-Date-2022-03-23_Time-17-00-14.log

<simpleError in (function (classes, fdef, mtable) {    methods <- .findInheritedMethods(classes, fdef, mtable)    if (length(methods) == 1L)         return(methods[[1L]])    else if (length(methods) == 0L) {        cnames <- paste0("\"", vapply(classes, as.character,             ""), "\"", collapse = ", ")        stop(gettextf("unable to find an inherited method for function %s for signature %s",             sQuote(fdef@generic), sQuote(cnames)), domain = NA)    }    else stop("Internal error in finding inherited methods; didn't return a unique method",         domain = NA)})(list("NULL"), new("standardGeneric", .Data = function (x) standardGeneric("seqnames"), generic = structure("seqnames", package = "GenomeInfoDb"),     package = "GenomeInfoDb", group = list(), valueClass = character(0),     signature = "x", default = NULL, skeleton = (function (x)     stop("invalid call in method dispatch to 'seqnames' (no default method)",         domain = NA))(x)), <environment>): unable to find an inherited method for function ‘seqnames’ for signature ‘"NULL"’>

************************************************************

2022-03-23 18:13:56 : (pbmc.6798.1x : 3 of 4) Adding GeneScoreMatrix!, 73.701 mins elapsed.

************************************************************
2022-03-23 18:14:00 : ERROR Found in .addGeneScoreMat for (pbmc.6798.1x : 3 of 4) 
LogFile = ArchRLogs/ArchR-createArrows-7ace23bf42f-Date-2022-03-23_Time-17-00-14.log

<simpleError in (function (classes, fdef, mtable) {    methods <- .findInheritedMethods(classes, fdef, mtable)    if (length(methods) == 1L)         return(methods[[1L]])    else if (length(methods) == 0L) {        cnames <- paste0("\"", vapply(classes, as.character,             ""), "\"", collapse = ", ")        stop(gettextf("unable to find an inherited method for function %s for signature %s",             sQuote(fdef@generic), sQuote(cnames)), domain = NA)    }    else stop("Internal error in finding inherited methods; didn't return a unique method",         domain = NA)})(list("NULL"), new("standardGeneric", .Data = function (x) standardGeneric("seqnames"), generic = structure("seqnames", package = "GenomeInfoDb"),     package = "GenomeInfoDb", group = list(), valueClass = character(0),     signature = "x", default = NULL, skeleton = (function (x)     stop("invalid call in method dispatch to 'seqnames' (no default method)",         domain = NA))(x)), <environment>): unable to find an inherited method for function ‘seqnames’ for signature ‘"NULL"’>

So, I feel like its not the problem of the prefix. But do not know why this happening either. Do you have any further comments?

Thanks

rcorces Mar 23, 2022
Maintainer

My mistake. Sorry for assuming it was a chr prefix issue.
I believe your geneAnnotation is not created correctly.

you've done the following, which basically adds the same GRanges object to all three of the params.

geneAnnotation <- createGeneAnnotation(
  TSS = gtf.gr, 
  exons = gtf.gr, 
  genes = gtf.gr)

That is not the correct way to do it. Perhaps looking at what a correct geneAnnotation looks like will help you (see below). You need to extract the exons and give those to exons, the single-base TSS regions and give those to TSS, and the genes and give those to genes.

> geneAnno <- getGeneAnnotation(projHeme5)
> geneAnno
List of length 3
names(3): genes exons TSS
> geneAnno$genes
GRanges object with 23274 ranges and 2 metadata columns:
          seqnames            ranges strand |     gene_id       symbol
             <Rle>         <IRanges>  <Rle> | <character>  <character>
      [1]     chr1       11874-14409      + |   100287102      DDX11L1
      [2]     chr1       14362-29961      - |      653635       WASH7P
      [3]     chr1       69091-70008      + |       79501        OR4F5
      [4]     chr1     134773-140566      - |      729737    LOC729737
      [5]     chr1     700245-714068      - |   100288069 LOC100288069
      ...      ...               ...    ... .         ...          ...
  [23270]     chrY 24455006-24462352      + |      159162      RBMY2FP
  [23271]     chrY 24455006-24564028      + |      159163       RBMY1F
  [23272]     chrY 24636544-24660784      + |        9081          PRY
  [23273]     chrY 25275502-25345254      - |        1617         DAZ1
  [23274]     chrY 25365581-27053187      + |       57055         DAZ2
  -------
  seqinfo: 24 sequences from hg19 genome
> geneAnno$exons
GRanges object with 687488 ranges and 2 metadata columns:
           seqnames            ranges strand |     gene_id      symbol
              <Rle>         <IRanges>  <Rle> | <character> <character>
       [1]     chr1       11874-12227      + |   100287102     DDX11L1
       [2]     chr1       11874-12227      + |   100287102     DDX11L1
       [3]     chr1       11874-12227      + |   100287102     DDX11L1
       [4]     chr1       12595-12721      + |   100287102     DDX11L1
       [5]     chr1       12613-12721      + |   100287102     DDX11L1
       ...      ...               ...    ... .         ...         ...
  [687484]     chrY 59338754-59338859      + |        3581        IL9R
  [687485]     chrY 59340194-59340278      + |        3581        IL9R
  [687486]     chrY 59340194-59340490      + |        3581        IL9R
  [687487]     chrY 59342487-59343488      + |        3581        IL9R
  [687488]     chrY 59342487-59343488      + |        3581        IL9R
  -------
  seqinfo: 24 sequences from hg19 genome
> geneAnno$TSS
GRanges object with 49052 ranges and 2 metadata columns:
          seqnames    ranges strand |     tx_id     tx_name
             <Rle> <IRanges>  <Rle> | <integer> <character>
      [1]     chr1     11874      + |         1  uc001aaa.3
      [2]     chr1     69091      + |         4  uc001aal.1
      [3]     chr1    321084      + |         5  uc001aaq.2
      [4]     chr1    321146      + |         6  uc001aar.2
      [5]     chr1    322037      + |         7  uc009vjk.2
      ...      ...       ...    ... .       ...         ...
  [49048]     chrY  27605678      - |     78803  uc004fwx.1
  [49049]     chrY  27606421      - |     78804  uc022cpc.1
  [49050]     chrY  27607432      - |     78805  uc004fwz.3
  [49051]     chrY  27635954      - |     78806  uc022cpd.1
  [49052]     chrY  59360854      - |     78807  uc011ncc.1
  -------
  seqinfo: 24 sequences from hg19 genome
>

HaixJiang · 2023-09-11T03:49:59Z

HaixJiang
Sep 11, 2023

Hi!
Have you solved your problem? Can you share it with me?

0 replies

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