create xmatrix and other issues

[JFerrer-B]

I'm very interested in implementing SCASA. I find it a very interesting method.
I'm trying to implement SCASA with gencode 44 as a reference. To do this, I'm trying to create the necessary files to be able to use this reference: Xmatrix, etc. To do this, I'm trying to replicate the algorithm explained at https://github.com/eudoraleer/scasa/wiki/How-to-run-Scasa-for-a-new-annotation

However, I haven't been able to fully execute it.

First, the Rscript code $scasaPath/gen_tx2gene.R is executed.
After executing this code, the following files are generated:
ref.sqlite
ref.tsv
ref.clean.fa
tx_removed.fa (the latter is not explained as far as I know and is the cause of an error in the code).
On line 106: names(tx.export.fasta)=paste0(names(tx.export.fasta)," ","Unknown-Gene-Name")
If all the cdna_tx are in genes.tx.all$TXNAME, it gives an error.
Also, line 95 is confusing:
cat("not in gtf: ",table(!p)) # some are not matched
If P has only one value and is TRUE, the cat returns as if all are not matched. It would be more understandable to use length(which(p==FALSE)).
I was able to resolve this error without any problems.

The problem comes with the following part of the code:
perl $scasaPath/SCRIPTS/SCASA_WRAP_V1.0.0.pl --createxmatrix YES --xmatrix $xmatrixFile --ref $refCleanFile --whitelist $whiteListFn

I get a lot of errors about not being able to create a folder that already exists, and it just gets stuck and doesn't move forward.

The error that appears is the following:

SCASA_WRAP_V1.0.0.pl --createxmatrix YES \
> --xmatrix $pwd/scasa_ref/Xmatrix.RData \
> --ref $pwd/scasa_ref/gencode.v44.transcripts.clean.fa \
> --whitelist $pwd/whitelist.txt

###############################################################
# 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 ./ already exists. Writing into existing directory..
mkdir: cannot create directory './/SCASA_My_Project_20250603141017/': File exists
Warning message:
In dir.create(output_dir, recursive = T) : 
'.//SCASA_My_Project_20250603141017/0PRESETS' already exists
Error in .Call2("C_solve_user_SEW", refwidths, start, end, width, translate.negative.coord, : 
row solving 16964: negative values ​​are not allowed in 'width'
Calls: simulate_experiment_countmat ... make_IRanges_from_windows_args -> solveUserSEW -> .Call2
Execution halted
ls: cannot access './/SCASA_My_Project_20250603141017/0PRESETS//SIMULATE_XMATRIX//*_1.fasta': No such file or directory

I hope you can answer my question.
Best regards,
Juan

[nghiavtr]

Hi Juan,

Thank you for reporting your issue of using Scasa.
As you mentioned, it is likely the issue mainly due to your run for gen_tx2gene.R file. It is not easy to know what made the bugs.

If you are using Gencode, you might consider to use the prebuilt annotation version 42 which is provided here: https://github.com/eudoraleer/scasa/tree/main/Anno

If you want to use Gencode version 44, you might try the following pipeline that I have successfully generated the annotation of version 42. (please replace the links to the files of version 42 by the corresponding ones for version 44)

Good lucks,
Nghia

##################
### require reformat.sh file from bbmap (https://sourceforge.net/projects/bbmap/)
# first, install BBMap and then set the path to the BBMap folder, so can run reformat.sh from any where:
wget https://deac-ams.dl.sourceforge.net/project/bbmap/BBMap_39.01.tar.gz
tar -xzf BBMap_39.01.tar.gz
export PATH=$PWD/bbmap/:$PATH 

# Download salmon alevin:
##################################################################
wget https://github.com/COMBINE-lab/salmon/releases/download/v1.4.0/salmon-1.4.0_linux_x86_64.tar.gz
tar -xzf salmon-1.4.0_linux_x86_64.tar.gz
export PATH=$PWD/salmon-latest_linux_x86_64/bin:$PATH
export LD_LIBRARY_PATH=$PWD/salmon-latest_linux_x86_64/lib:$LD_LIBRARY_PATH

#NOTE: in some linux versions, we need to remove libm.so.6 which make a conflict with bbmap
rm $PWD/salmon-latest_linux_x86_64/lib/libm.so.6

#download annotation and unzip
wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_42/gencode.v42.transcripts.fa.gz
wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_42/gencode.v42.annotation.gtf.gz
gunzip gencode.v42.annotation.gtf.gz
gunzip gencode.v42.transcripts.fa.gz

#download whiteList file
wget  https://github.com/10XGenomics/cellranger/raw/master/lib/python/cellranger/barcodes/3M-february-2018.txt.gz
gunzip 3M-february-2018.txt.gz

#download scasa from github
git clone https://github.com/eudoraleer/scasa.git

export PATH=$PWD/bbmap/:$PATH
export PATH=$PWD/salmon-latest_linux_x86_64/bin:$PATH
export LD_LIBRARY_PATH=$PWD/salmon-latest_linux_x86_64/lib:$LD_LIBRARY_PATH

scasaPath=$PWD/scasa/scasa
export PATH=$scasaPath:$PATH # for scasa

whiteListFn=$PWD/3M-february-2018.txt
refFile=$PWD/gencode.v42.transcripts.fa
gtfFile=$PWD/gencode.v42.annotation.gtf
sqliteFile=$PWD/gencode.v42.sqlite
txp2geneFile=$PWD/gencode.v42.tsv
refCleanFile=$PWD/gencode.v42.clean.fa
xmatrixFile=$PWD/Xmatrix.RData #fake and redundant Xmatrix file to run scasa, which does not exist. WILL FIX THIS SOON
isoformMapperFile=$PWD/isoform_groups_gencode.v42_mapper.RData

# create txp2gene file and clean the cdna to keep only transcripts existing in gtf annotation. The refCleanFile will be used for downstream analysis
Rscript $scasaPath/../aux/gen_tx2gene.R cdna=$refFile gtf=$gtfFile anntype=GENCODE sqlite=$sqliteFile cdnaout=$refCleanFile out=$txp2geneFile

#update txp2geneFile in the scasa/REFERENCE/ (DO IT BEFORE the step of creating X-matrix)
cp $txp2geneFile $scasaPath/REFERENCE/txp2gene_hg38.tsv

#create X-matrix
#scasa --createxmatrix YES --xmatrix $xmatrixFile --ref $refCleanFile --whitelist $whiteListFn
perl $scasaPath/SCRIPTS/SCASA_WRAP_V1.0.0.pl --createxmatrix YES --xmatrix $xmatrixFile --ref $refCleanFile --whitelist $whiteListFn


#create isoformMapper
newxmatrixFile=$PWD/SCASA_My_Project_*/0PRESETS/SIMULATE_XMATRIX/Xmatrix.RData
newxmatrixFile=($newxmatrixFile)
newxmatrixFile=${newxmatrixFile[0]}
Rscript $scasaPath/../aux/categorise_isoforms.R sqlite=$sqliteFile xmatrix=$newxmatrixFile out=$isoformMapperFile


mkdir OUTPUT
cp $newxmatrixFile OUTPUT/X_matrix.RData
cp $isoformMapperFile OUTPUT/isoformMapper.RData
cp $txp2geneFile OUTPUT/txp2gene.tsv
cp $refCleanFile OUTPUT/transcripts_clean.fa