Alignment of multiple files using STARSolo

Question

[otieno43]

I am trying to align single cell RNA-data set using using STAR and following the manual. I however can't get this to run.

STAR --runThreadN 10 --genomeDir ./T_congoSc/Tcongo_noUTR-index \
        --readFilesIn ./Tcongo_scell/Tcongo_L002_R2_001.fastq.gz,Tcongo_L003_R2_001.fastq.gz ./Tcongo_scell/Tcongo_L002_R1_001.fastq.gz,Tcongo_L003_R1_001.fastq.gz \
        --outFilterScoreMin 30 --clipAdapterType CellRanger4 --soloType CB_UMI_Simple --soloCBstart 1 --soloUMIstart 17 --soloCBlen 16 --soloUMIlen 12 --soloBarcodeReadLength 0 \
        --soloCBwhitelist ./barcodes/3M-february-2018.txt --soloUMIdedup 1MM_CR \
        --soloCBmatchWLtype 1MM_multi_Nbase_pseudocounts --soloUMIfiltering MultiGeneUMI_CR \
        --outFileNamePrefix  ./mapping_stats/Tcongo_2 \
        --soloMultiMappers EM --soloFeatures Gene --readFilesCommand zcat --soloCellFilter  EmptyDrops_CR

It does not seem to recognize Tcongo_L003_R2_001.fastq.gz and Tcongo_L003_R1_001.fastq.gz files. When I tried to give paths to each of these, I get the same error below.

!!!!! WARNING:  Could not ls Tcongo_1_TCO_S1_L003_R2_001.fastq.gz

EXITING: because of fatal INPUT file error: could not open read file: Tcongo_1_TCO_S1_L003_R2_001.fastq.gz
SOLUTION: check that this file exists and has read permision.

Any suggestion please.

[biofalconch]

It would be good if you tell us if you had checked if these files exist, maybe they are in another directory? Could you post the output of ls -l ./Tcongo_scell/ ?

[otieno43]

The files exist,

ls -lah
-rw-r--r-- 1 ea432 1.1G May 27  2023 Tcongo_L002_I1_001.fastq.gz
-rw-r--r-- 1 ea432 1.1G May 27  2023 Tcongo_L002_R1_001.fastq.gz
-rw-r--r-- 1 ea432 1.1G May 27  2023 Tcongo_L002_R2_001.fastq.gz
-rw-r--r-- 1 ea432 1.1G May 27  2023 Tcongo_L003_I1_001.fastq.gz
-rw-r--r-- 1 ea432 3.6G May 27  2023 Tcongo_L003_R1_001.fastq.gz
-rw-r--r-- 1 ea432 8.1G May 27  2023 Tcongo_L003_R2_001.fastq.gz

Answer 1

[Jack Tierney]

You need to provide the path to each read file. Not just the name.

Provide --readFilesIn ./Tcongo_scell/Tcongo_L002_R2_001.fastq.gz,./Tcongo_scell/Tcongo_L003_R2_001.fastq.gz instead of ./Tcongo_scell/Tcongo_L002_R2_001.fastq.gz,Tcongo_L003_R2_001.fastq.gz

Full Command:

STAR --runThreadN 10 --genomeDir ./T_congoSc/Tcongo_noUTR-index \
        --readFilesIn ./Tcongo_scell/Tcongo_L002_R2_001.fastq.gz,./Tcongo_scell/Tcongo_L003_R2_001.fastq.gz ./Tcongo_scell/Tcongo_L002_R1_001.fastq.gz,./Tcongo_scell/Tcongo_L003_R1_001.fastq.gz \
        --outFilterScoreMin 30 --clipAdapterType CellRanger4 --soloType CB_UMI_Simple --soloCBstart 1 --soloUMIstart 17 --soloCBlen 16 --soloUMIlen 12 --soloBarcodeReadLength 0 \
        --soloCBwhitelist ./barcodes/3M-february-2018.txt --soloUMIdedup 1MM_CR \
        --soloCBmatchWLtype 1MM_multi_Nbase_pseudocounts --soloUMIfiltering MultiGeneUMI_CR \
        --outFileNamePrefix  ./mapping_stats/Tcongo_2 \
        --soloMultiMappers EM --soloFeatures Gene --readFilesCommand zcat --soloCellFilter  EmptyDrops_CR

[otieno43]

Hi Jack, I did provide the full command or path, it still did not work.

[GenoMax]

Did you provide full path for each file in the list. You can't do that for just one initial file.

[otieno43]

Thanks. It is working now.