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Error while excuting emptydrops #346

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loguille opened this issue Jul 17, 2024 · 1 comment
Open

Error while excuting emptydrops #346

loguille opened this issue Jul 17, 2024 · 1 comment
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bug Something isn't working

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@loguille
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Description of the bug

The error appear when nf-core/scrnaseq is launched with any aligner at the emptydrop step. The pipeline is labelled as completed but with errors as it failed with the output error : "Only kallisto and alevin have transposed matrices."

Command used and terminal output

nextflow run nf-core/scrnaseq    -profile docker  --fasta https://github.com/nf-core/test-datasets/raw/scrnaseq/reference/GRCm38.p6.genome.chr19.fa --gtf  https://github.com/nf-core/test-datasets/raw/scrnaseq/reference/gencode.vM19.annotation.chr19.gtf   --input samplesheet.csv --protocol 10XV2  --aligner alevin  --outdir alignment_results_pbmc/  --max_memory 24.GB

executor >  local (8)
[1c/73abea] NFC…E_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (PBMC_10K) | 1 of 1 ✔
[14/7bbcd5] NFC…RNASEQ:GTF_GENE_FILTER (GRCm38.p6.genome.chr19.fa) | 1 of 1 ✔
[a1/972267] NFC…:SIMPLEAF_INDEX (GRCm38.p6.genome.chr19_genes.gtf) | 1 of 1 ✔
[07/7e0538] NFC…SCRNASEQ:SCRNASEQ_ALEVIN:SIMPLEAF_QUANT (PBMC_10K) | 1 of 1 ✔
[4e/cbb7d5] NFC…NASEQ:SCRNASEQ:SCRNASEQ_ALEVIN:ALEVINQC (PBMC_10K) | 1 of 1 ✔
[08/2d886b] NFC…RNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (PBMC_10K) | 1 of 1, failed: 1 ✘
[cd/660df7] NFC…SEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (PBMC_10K) | 1 of 1 ✔
[-        ] NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD    -
[-        ] NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT  | 0 of 1
[fd/9a5554] NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                       | 1 of 1 ✔
Execution cancelled -- Finishing pending tasks before exit
-[nf-core/scrnaseq] Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (PBMC_10K)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (PBMC_10K)` terminated with an error exit status (1)


Command executed:

  mkdir emptydrops_filtered/
  emptydrops_cell_calling.R \
      *_alevin_results/af_quant/alevin/quants_mat.mtx \
      *_alevin_results/af_quant/alevin/quants_mat_rows.txt \
      *_alevin_results/af_quant/alevin/quants_mat_cols.txt \
      emptydrops_filtered \
      alevin \
      0

Command exit status:
  1

Command output:
  [1] "Only kallisto and alevin have transposed matrices."

Command error:
  
  The following objects are masked from ‘package:base’:
  
      anyDuplicated, aperm, append, as.data.frame, basename, cbind,
      colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
      get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
      match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
      Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
      table, tapply, union, unique, unsplit, which.max, which.min
  
  Loading required package: S4Vectors
  
  Attaching package: ‘S4Vectors’
  
  The following objects are masked from ‘package:base’:
  
      expand.grid, I, unname
  
  Loading required package: IRanges
  Loading required package: GenomeInfoDb
  Loading required package: Biobase
  Welcome to Bioconductor
  
      Vignettes contain introductory material; view with
      'browseVignettes()'. To cite Bioconductor, see
      'citation("Biobase")', and for packages 'citation("pkgname")'.
  
  
  Attaching package: ‘Biobase’
  
  The following object is masked from ‘package:MatrixGenerics’:
  
      rowMedians
  
  The following objects are masked from ‘package:matrixStats’:
  
      anyMissing, rowMedians
  
  
  Attaching package: ‘Matrix’
  
  The following object is masked from ‘package:S4Vectors’:
  
      expand
  
  [1] "Only kallisto and alevin have transposed matrices."
  Error in .local(m, ...) : 
    insufficient unique points for computing knee/inflection points
  Calls: emptyDrops ... .local -> do.call -> <Anonymous> -> <Anonymous> -> .local
  Execution halted

Work dir:
  /home/loic/UCL_work/pipeline_scRNAseq/work/08/2d886bcd7288f56e8fc4a99b34dedd

Tip: when you have fixed the problem you can continue the execution adding the option `-resume` to the run command line

 -- Check '.nextflow.log' file for details
ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting

 -- Check '.nextflow.log' file for details

Relevant files

nextflow.log

System information

Nextflow version 24.04.3
Hardware Desktop
Executor local
Container engine Docker
Os Ubuntu
Version of nf-core/scrnaseq v2.7.0
@loguille loguille added the bug Something isn't working label Jul 17, 2024
@YaoLei-Leo
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I met the similar error today.

(scRNA) leiyao@ProfWLYangLabServer:/mnt/Extend1/leiyao/20240716_JN_SLEscRNA$ bash 01.test.sh 

 N E X T F L O W   ~  version 24.04.3

Launching `https://github.com/nf-core/scrnaseq` [compassionate_borg] DSL2 - revision: c8f02f515d [master]



------------------------------------------------------
                                        ,--./,-.
        ___     __   __   __   ___     /,-._.--~'
  |\ | |__  __ /  ` /  \ |__) |__         }  {
  | \| |       \__, \__/ |  \ |___     \`-._,-`-,
                                        `._,._,'
  nf-core/scrnaseq v2.7.0-gc8f02f5
------------------------------------------------------
Core Nextflow options
  revision       : master
  runName        : compassionate_borg
  containerEngine: singularity
  launchDir      : /mnt/Extend1/leiyao/20240716_JN_SLEscRNA
  workDir        : /mnt/Extend1/leiyao/20240716_JN_SLEscRNA/work
  projectDir     : /home/leiyao/.nextflow/assets/nf-core/scrnaseq
  userName       : leiyao
  profile        : singularity
  configFiles    : 

Input/output options
  input          : samplesheet.csv
  outdir         : ./Result

Mandatory arguments
  aligner        : cellranger
  protocol       : 10XV2

Reference genome options
  fasta          : ./Data/Homo_sapiens.GRCh38.dna.toplevel.fa
  gtf            : ./Data/Homo_sapiens.GRCh38.112.gtf

!! Only displaying parameters that differ from the pipeline defaults !!
------------------------------------------------------
If you use nf-core/scrnaseq for your analysis please cite:

* The pipeline
  https://doi.org/10.5281/zenodo.3568187

* The nf-core framework
  https://doi.org/10.1038/s41587-020-0439-x

* Software dependencies
  https://github.com/nf-core/scrnaseq/blob/master/CITATIONS.md
------------------------------------------------------
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC               -
executor >  local (1)
executor >  local (2)
executor >  local (5)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
executor >  local (5)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
executor >  local (9)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
[12/ed39cd] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (Homo_sapiens.GRCh38.dna.toplevel.fa)                          [100%] 1 of 1 ✔
[9a/ac459f] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKGTF (Homo_sapiens.GRCh38.dna.toplevel_genes.gtf) [100%] 1 of 1 ✔
[ac/24f578] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKREF (Homo_sapiens.GRCh38.dna.toplevel.fa)        [100%] 1 of 1 ✔
[77/62a987] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT (L01_3)                                      [100%] 1 of 1 ✔
[21/06b153] process > NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)                                                [  0%] 0 of 1
[46/3f7020] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (L01_3)                                             [100%] 2 of 2
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                                                     -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                                                   -
[84/804f99] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                                                        [100%] 1 of 1 ✔
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)` terminated with an error exit status (1)


Command executed:

  mkdir emptydrops_filtered/
  emptydrops_cell_calling.R \
      matrix.mtx.gz \
      barcodes.tsv.gz \
      features.tsv.gz \
      emptydrops_filtered \
      cellranger \
      0

Command exit status:
  1

Command output:
  [1] "Only kallisto and alevin have transposed matrices."

Command error:
  
  The following objects are masked from ‘package:base’:
  
      anyDuplicated, aperm, append, as.data.frame, basename, cbind,
      colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
      get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
      match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
      Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
      table, tapply, union, unique, unsplit, which.max, which.min
  
  Loading required package: S4Vectors
  
  Attaching package: ‘S4Vectors’
  
  The following objects are masked from ‘package:base’:
  
      expand.grid, I, unname
  
  Loading required package: IRanges
  Loading required package: GenomeInfoDb
  Loading required package: Biobase
  Welcome to Bioconductor
  
      Vignettes contain introductory material; view with
      'browseVignettes()'. To cite Bioconductor, see
      'citation("Biobase")', and for packages 'citation("pkgname")'.
executor >  local (9)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
[12/ed39cd] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (Homo_sapiens.GRCh38.dna.toplevel.fa)                          [100%] 1 of 1 ✔
[9a/ac459f] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKGTF (Homo_sapiens.GRCh38.dna.toplevel_genes.gtf) [100%] 1 of 1 ✔
[ac/24f578] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKREF (Homo_sapiens.GRCh38.dna.toplevel.fa)        [100%] 1 of 1 ✔
[77/62a987] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT (L01_3)                                      [100%] 1 of 1 ✔
[21/06b153] process > NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)                                                [100%] 1 of 1, failed: 1 ✘
[46/3f7020] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (L01_3)                                             [100%] 2 of 2 ✔
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                                                     -
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                                                   -
[84/804f99] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                                                        [100%] 1 of 1 ✔
Execution cancelled -- Finishing pending tasks before exit
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)` terminated with an error exit status (1)


Command executed:

  mkdir emptydrops_filtered/
  emptydrops_cell_calling.R \
      matrix.mtx.gz \
      barcodes.tsv.gz \
      features.tsv.gz \
      emptydrops_filtered \
      cellranger \
      0

Command exit status:
  1

Command output:
  [1] "Only kallisto and alevin have transposed matrices."

Command error:
  
  The following objects are masked from ‘package:base’:
  
      anyDuplicated, aperm, append, as.data.frame, basename, cbind,
      colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
      get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
      match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
      Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
      table, tapply, union, unique, unsplit, which.max, which.min
  
  Loading required package: S4Vectors
  
  Attaching package: ‘S4Vectors’
  
  The following objects are masked from ‘package:base’:
  
      expand.grid, I, unname
  
  Loading required package: IRanges
  Loading required package: GenomeInfoDb
  Loading required package: Biobase
  Welcome to Bioconductor
  
      Vignettes contain introductory material; view with
      'browseVignettes()'. To cite Bioconductor, see
      'citation("Biobase")', and for packages 'citation("pkgname")'.
  
  
  Attaching package: ‘Biobase’
executor >  local (9)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
[12/ed39cd] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (Homo_sapiens.GRCh38.dna.toplevel.fa)                          [100%] 1 of 1 ✔
[9a/ac459f] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKGTF (Homo_sapiens.GRCh38.dna.toplevel_genes.gtf) [100%] 1 of 1 ✔
[ac/24f578] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKREF (Homo_sapiens.GRCh38.dna.toplevel.fa)        [100%] 1 of 1 ✔
[77/62a987] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT (L01_3)                                      [100%] 1 of 1 ✔
[21/06b153] process > NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)                                                [100%] 1 of 1, failed: 1 ✘
[46/3f7020] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (L01_3)                                             [100%] 2 of 2 ✔
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                                                     [  0%] 0 of 2
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                                                   -
[84/804f99] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                                                        [100%] 1 of 1 ✔
Execution cancelled -- Finishing pending tasks before exit
-[nf-core/scrnaseq] Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)` terminated with an error exit status (1)


Command executed:

  mkdir emptydrops_filtered/
  emptydrops_cell_calling.R \
      matrix.mtx.gz \
      barcodes.tsv.gz \
      features.tsv.gz \
      emptydrops_filtered \
      cellranger \
      0

Command exit status:
  1

Command output:
  [1] "Only kallisto and alevin have transposed matrices."

Command error:
  
  The following objects are masked from ‘package:base’:
  
      anyDuplicated, aperm, append, as.data.frame, basename, cbind,
      colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
      get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
      match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
      Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
      table, tapply, union, unique, unsplit, which.max, which.min
  
  Loading required package: S4Vectors
  
  Attaching package: ‘S4Vectors’
  
  The following objects are masked from ‘package:base’:
  
      expand.grid, I, unname
  
  Loading required package: IRanges
  Loading required package: GenomeInfoDb
  Loading required package: Biobase
  Welcome to Bioconductor
  
      Vignettes contain introductory material; view with
      'browseVignettes()'. To cite Bioconductor, see
      'citation("Biobase")', and for packages 'citation("pkgname")'.
  
  
  Attaching package: ‘Biobase’
executor >  local (9)
[9c/23caad] process > NFCORE_SCRNASEQ:SCRNASEQ:FASTQC_CHECK:FASTQC (L01_3)                                                    [100%] 1 of 1 ✔
[12/ed39cd] process > NFCORE_SCRNASEQ:SCRNASEQ:GTF_GENE_FILTER (Homo_sapiens.GRCh38.dna.toplevel.fa)                          [100%] 1 of 1 ✔
[9a/ac459f] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKGTF (Homo_sapiens.GRCh38.dna.toplevel_genes.gtf) [100%] 1 of 1 ✔
[ac/24f578] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_MKREF (Homo_sapiens.GRCh38.dna.toplevel.fa)        [100%] 1 of 1 ✔
[77/62a987] process > NFCORE_SCRNASEQ:SCRNASEQ:CELLRANGER_ALIGN:CELLRANGER_COUNT (L01_3)                                      [100%] 1 of 1 ✔
[21/06b153] process > NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)                                                [100%] 1 of 1, failed: 1 ✘
[46/3f7020] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_H5AD (L01_3)                                             [100%] 2 of 2 ✔
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:CONCAT_H5AD                                                     [  0%] 0 of 2
[-        ] process > NFCORE_SCRNASEQ:SCRNASEQ:MTX_CONVERSION:MTX_TO_SEURAT                                                   -
[84/804f99] process > NFCORE_SCRNASEQ:SCRNASEQ:MULTIQC                                                                        [100%] 1 of 1 ✔
Execution cancelled -- Finishing pending tasks before exit
-[nf-core/scrnaseq] Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)'

Caused by:
  Process `NFCORE_SCRNASEQ:SCRNASEQ:EMPTYDROPS_CELL_CALLING (L01_3)` terminated with an error exit status (1)


Command executed:

  mkdir emptydrops_filtered/
  emptydrops_cell_calling.R \
      matrix.mtx.gz \
      barcodes.tsv.gz \
      features.tsv.gz \
      emptydrops_filtered \
      cellranger \
      0

Command exit status:
  1

Command output:
  [1] "Only kallisto and alevin have transposed matrices."

Command error:
  
  The following objects are masked from ‘package:base’:
  
      anyDuplicated, aperm, append, as.data.frame, basename, cbind,
      colnames, dirname, do.call, duplicated, eval, evalq, Filter, Find,
      get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply,
      match, mget, order, paste, pmax, pmax.int, pmin, pmin.int,
      Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
      table, tapply, union, unique, unsplit, which.max, which.min
  
  Loading required package: S4Vectors
  
  Attaching package: ‘S4Vectors’
  
  The following objects are masked from ‘package:base’:
  
      expand.grid, I, unname
  
  Loading required package: IRanges
  Loading required package: GenomeInfoDb
  Loading required package: Biobase
  Welcome to Bioconductor
  
      Vignettes contain introductory material; view with
      'browseVignettes()'. To cite Bioconductor, see
      'citation("Biobase")', and for packages 'citation("pkgname")'.
  
  
  Attaching package: ‘Biobase’
  
  The following object is masked from ‘package:MatrixGenerics’:
  
      rowMedians
  
  The following objects are masked from ‘package:matrixStats’:
  
      anyMissing, rowMedians
  
  
  Attaching package: ‘Matrix’
  
  The following object is masked from ‘package:S4Vectors’:
  
      expand
  
  [1] "Only kallisto and alevin have transposed matrices."
  Error in .local(m, ...) : 
    insufficient unique points for computing knee/inflection points
  Calls: emptyDrops ... .local -> do.call -> <Anonymous> -> <Anonymous> -> .local
  Execution halted

Work dir:
  /mnt/Extend1/leiyao/20240716_JN_SLEscRNA/work/21/06b153ef96a6515ec7073a1c6773c6

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.sh`

 -- Check '.nextflow.log' file for details
ERROR ~ Pipeline failed. Please refer to troubleshooting docs: https://nf-co.re/docs/usage/troubleshooting

 -- Check '.nextflow.log' file for details

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@loguille @YaoLei-Leo