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eudoraleer / scasa

SCASA: Single cell transcript quantification tool
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Using Drosophila scRNAseq doesn't work. #17

Open enguyen117 opened 2 weeks ago

enguyen117 commented 2 weeks ago 

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                     You are running Scasa v1.0.1 using docker ....
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Loading parameters from file...

##############################################################
#       SCASA V1.0.1
#       SINGLE CELL TRANSCRIPT QUANTIFICATION TOOL
#       Version Date: 2022-03-24
#       FOR ANY ISSUES, CONTACT: LU.PAN@KI.SE
#       https://github.com/eudoraleer/scasa/
##############################################################

Directory /source/output already exists. Writing into existing directory..
mkdir: cannot create directory '/source/output/SCASA_My_Project_20241018115607/': File exists

Preparing for alignment..
Indexing reference..
Directory /source/output/SCASA_My_Project_20241018115607/0PRESETS//REF_INDEX/ already exists. Writing into existing directory..
Version Info: ### PLEASE UPGRADE SALMON ###
### A newer version of salmon with important bug fixes and improvements is available. ####
###
The newest version, available at https://github.com/COMBINE-lab/salmon/releases
contains new features, improvements, and bug fixes; please upgrade at your
earliest convenience.
###
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###[2024-10-18 11:56:08.181] [jLog] [warning] The salmon index is being built without any decoy sequences.  It is recommended that decoy sequence (either computed auxiliary decoy sequence or the genome of the organism) be provided during indexing. Further details can be found at https://salmon.readthedocs.io/en/latest/salmon.html#preparing-transcriptome-indices-mapping-based-mode.
[2024-10-18 11:56:08.181] [jLog] [info] building index
out : /source/output/SCASA_My_Project_20241018115607/0PRESETS//REF_INDEX/
[2024-10-18 11:56:08.183] [puff::index::jointLog] [info] Running fixFasta

[Step 1 of 4] : counting k-mers
[2024-10-18 11:56:08.198] [puff::index::jointLog] [warning] Entry with header [:1865449_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.198] [puff::index::jointLog] [warning] Entry with header [:1865432_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.199] [puff::index::jointLog] [warning] Entry with header [:1865325_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.199] [puff::index::jointLog] [warning] Entry with header [:1865381_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.252] [puff::index::jointLog] [warning] Entry with header [:1831393_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.281] [puff::index::jointLog] [warning] Entry with header [:1832312_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.284] [puff::index::jointLog] [warning] Entry with header [:1832497_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.286] [puff::index::jointLog] [warning] Entry with header [:1832637_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.369] [puff::index::jointLog] [warning] Entry with header [:1713479_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.370] [puff::index::jointLog] [warning] Entry with header [:1713453_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.378] [puff::index::jointLog] [warning] Entry with header [:1713664_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.402] [puff::index::jointLog] [warning] Entry with header [:1930839_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.402] [puff::index::jointLog] [warning] Entry with header [:1930954_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.402] [puff::index::jointLog] [warning] Entry with header [:1930872_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.402] [puff::index::jointLog] [warning] Entry with header [:1930893_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.404] [puff::index::jointLog] [warning] Entry with header [:1931167_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.407] [puff::index::jointLog] [warning] Entry with header [:1931119_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.407] [puff::index::jointLog] [warning] Entry with header [:1931073_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.408] [puff::index::jointLog] [warning] Entry with header [:1931092_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.410] [puff::index::jointLog] [warning] Entry with header [:1931418_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.410] [puff::index::jointLog] [warning] Entry with header [:1931431_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.461] [puff::index::jointLog] [warning] Entry with header [:236488_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:08.468] [puff::index::jointLog] [warning] Entry with header [:1932286_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.290] [puff::index::jointLog] [warning] Entry with header [:366811_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.382] [puff::index::jointLog] [warning] Entry with header [:342371_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.453] [puff::index::jointLog] [warning] Entry with header [:368993_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.467] [puff::index::jointLog] [warning] Entry with header [:908763_sim4], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.480] [puff::index::jointLog] [warning] Entry with header [:368945_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)

...

[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077660_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077726_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077727_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077732_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077743_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077749_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077759_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077773_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077806_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077835_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077837_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.848] [puff::index::jointLog] [warning] Entry with header [proteomic_PAp00077838_HIT], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.913] [puff::index::jointLog] [warning] Entry with header [:1125343_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:09.986] [puff::index::jointLog] [warning] Discarding entry with header [:1178069_sim4], since it had length 0 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.018] [puff::index::jointLog] [warning] Discarding entry with header [:1178074_sim4], since it had length 0 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.018] [puff::index::jointLog] [warning] Discarding entry with header [:1178076_sim4], since it had length 0 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.148] [puff::index::jointLog] [warning] Entry with header [:1128697_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.160] [puff::index::jointLog] [warning] Entry with header [:1122427_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.302] [puff::index::jointLog] [warning] Entry with header [:1123735_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.308] [puff::index::jointLog] [warning] Entry with header [:1124142_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.315] [puff::index::jointLog] [warning] Entry with header [:1124366_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.457] [puff::index::jointLog] [warning] Entry with header [:1171695_blastx_masked], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.476] [puff::index::jointLog] [warning] Entry with header [:1123831_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.477] [puff::index::jointLog] [warning] Entry with header [:1123807_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.483] [puff::index::jointLog] [warning] Entry with header [:1127151_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.488] [puff::index::jointLog] [warning] Entry with header [:1128407_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.488] [puff::index::jointLog] [warning] Entry with header [:1128413_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.523] [puff::index::jointLog] [warning] Entry with header [:346384_repeatmasker] was longer than 400000 nucleotides.  This is probably a chromosome instead of a transcript.
[2024-10-18 11:56:10.528] [puff::index::jointLog] [warning] Entry with header [:1170614_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.531] [puff::index::jointLog] [warning] Entry with header [:352261_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.553] [puff::index::jointLog] [warning] Entry with header [:375430_repeatmasker] was longer than 400000 nucleotides.  This is probably a chromosome instead of a transcript.
[2024-10-18 11:56:10.560] [puff::index::jointLog] [warning] Entry with header [:376441_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.582] [puff::index::jointLog] [warning] Entry with header [:346525_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.594] [puff::index::jointLog] [warning] Entry with header [:349880_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.605] [puff::index::jointLog] [warning] Entry with header [:359995_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.622] [puff::index::jointLog] [warning] Entry with header [:355395_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.643] [puff::index::jointLog] [warning] Entry with header [:362295_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.645] [puff::index::jointLog] [warning] Entry with header [:375072_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.646] [puff::index::jointLog] [warning] Entry with header [:366667_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.647] [puff::index::jointLog] [warning] Entry with header [:349820_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.648] [puff::index::jointLog] [warning] Entry with header [:347269_repeatmasker], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.687] [puff::index::jointLog] [warning] Entry with header [:978794_sim4], had length less than equal to the k-mer length of 31 (perhaps after poly-A clipping)
[2024-10-18 11:56:10.689] [puff::index::jointLog] [error] In FixFasta, two references with the same name but different sequences: :516332_sim4. We require that all input records have a unique name up to the first whitespace (or user-provided separator) character.
Finnished indexing reference..
Begins pseudo-alignment..
nohup: redirecting stderr to stdout
Congratulations! Pseudo-alignment has completed in 30 seconds!
Scasa quantification has started..
Begin Scasa quantification for sample FCA34_Male_heart_adult_5dWT_Bodmer_sample1_S19_L002..
Error in file(con, "r") : cannot open the connection
Calls: readLines -> file
In addition: Warning message:
In file(con, "r") :
  cannot open file '/source/output/SCASA_My_Project_20241018115607/1ALIGN//FCA34_Male_heart_adult_5dWT_Bodmer_sample1_S19_L002_alignout/alevin/bfh.txt': No such file or directory
Execution halted
Loading required package: iterators
Loading required package: parallel
Error in readChar(con, 5L, useBytes = TRUE) : cannot open the connection
Calls: load -> readChar
In addition: Warning message:
In readChar(con, 5L, useBytes = TRUE) :
  cannot open compressed file '/source/output/SCASA_My_Project_20241018115607/2QUANT/FCA34_Male_heart_adult_5dWT_Bodmer_sample1_S19_L002_quant/Sample_eqClass.RData', probable reason 'No such file or directory'
Execution halted
Error in readChar(con, 5L, useBytes = TRUE) : cannot open the connection
Calls: load -> readChar
In addition: Warning message:
In readChar(con, 5L, useBytes = TRUE) :
  cannot open compressed file '/source/output/SCASA_My_Project_20241018115607/2QUANT//FCA34_Male_heart_adult_5dWT_Bodmer_sample1_S19_L002_quant//scasa_isoform_expression.RData', probable reason 'No such file or directory'
Execution halted
Congratulations! Scasa single cell RNA-Seq transcript quantification has completed in 30 seconds!
All done!

Hi! I've used scasa before and published work on it. I am trying to use scasa with drosophila data. I used the drosophila new annotation you guys did here: https://github.com/eudoraleer/scasa/wiki/How-to-run-Scasa-for-a-new-annotation. I am using this reference genome: dmel-all-aligned-r6.46. I also am using the Docker version of scasa. But I keep running into this error. Scasa works fine when I do the sample case with 200 cells. Any help would be great!

nghiavtr commented 1 week ago

Hi @enguyen117 From the log information, there was something wrong with the reference that you used to index:

_[2024-10-18 11:56:10.689] [puff::index::jointLog] [error] In FixFasta, two references with the same name but different sequences: :516332sim4. We require that all input records have a unique name up to the first whitespace (or user-provided separator) character.

You have two transcripts with the same names in the reference, please fix their names. Also, when you run Scasa for annotation not from Human, please follow the instruction and do not use the docker version.

Nghia