Empty output

[MJ-1987]

Hi, although the 1.4.1 version is now running without errors for the chromosome naming, unfortunately, the output is empty for all 3 files that were generated
This is the log file without any errors.
Any idea what happened?

scReadCounts Options:
SNV Files (-s): cDNA3_SNP_dedup.vcf
Read Files (-r): bam-files/cDNA3_haplotagged_pacbio_sorted.bam
Cell Barcode (-C): CellRanger
Description:
Cell barcodes from the CB tag of aligned read -
reads without a CB tag or with CB tag not in the
accept list (default: file "barcodes.tsv" in the
current directory) dropped.
Specification:
tag=CB
acceptlist=barcodes.tsv
UMI Count (-U): None
Read/Alignment Filter (-f): Basic
Description:
Filter alignments flagged as SECONDARY, DUPLICATE,
UNMAPPED, or QCFAIL, and those with a gap or indel
at SNV locus - minimal recommended filtering
strategy.
Specification:
skip_duplicate=True
skip_secondary=True
skip_qcfail=True
skip_unmapped=True
Outfile File (-o): cDN3_pacbio_64_out.txt

Advanced:
Min. Reads (-m) 5 (applied only to VAF, FVAF, RVAF)
Max. Reads (-M): None
Directional Counts (-D): False
Valid Cell Barcode (-b): None
Threads (-t): 64
Force (-F): False
Quiet (-q): False

Command-Line: scReadCounts -s "cDNA3_SNP_dedup.vcf" -r "bam-files/cDNA3_haplotagged_pacbio_sorted.bam" -C "CellRanger" -t 64 -o "cDN3_pacbio_64_out.txt"

Execute readCounts...

readCounts Options:
SNV Files (-s): cDNA3_SNP_dedup.vcf
Read Files (-r): bam-files/cDNA3_haplotagged_pacbio_sorted.bam
Read/Alignment Filter (-f): Basic
Description:
Filter alignments flagged as SECONDARY, DUPLICATE,
UNMAPPED, or QCFAIL, and those with a gap or indel
at SNV locus - minimal recommended filtering
strategy.
Specification:
skip_duplicate=True
skip_secondary=True
skip_qcfail=True
skip_unmapped=True
Outfile File (-o): cDN3_pacbio_64_out.txt

Advanced:
Min. Reads (-m) 0
Max. Reads (-M): None
SNV Batch Size (-B):
Read Groups (-G): CellRanger_CB
Description:
Cell barcodes from the CB tag of aligned read -
reads without a CB tag or with CB tag not in the
accept list (default: file "barcodes.tsv" in the
current directory) dropped.
Specification:
tag=CB
acceptlist=barcodes.tsv
Valid Read Groups (-b):
UMI Count (-U): None
Threads (-t): 64
Extended Output (-E): None
Quiet (-q): False

Command-Line: readCounts -s "cDNA3_SNP_dedup.vcf" -r "bam-files/cDNA3_haplotagged_pacbio_sorted.bam" -m 0 -G "CellRanger_CB" -t 64 -o "cDN3_pacbio_64_out.txt"

Read SNV data ->|
************************************************************ (2:41 min)
SNVs: 6154117
Count reads per SNV ->|
************************************************************ (18:25:19 hrs)
SNVs/sec: 92.80
Output results (0.01 sec)

Execute readCountsMatrix for Ref;Var matrix...

readCountsMatrix Options:
ReadCounts Files (-c): cDN3_pacbio_64_out.txt
Matrix Output (-M): Ref;Var
Min. Reads (-m): 0
Quiet (-q): False
Outfile File (-o): cDN3_pacbio_64_out.cnt.matrix.txt

Command-Line: readCountsMatrix -c "cDN3_pacbio_64_out.txt" -M Ref;Var -o "cDN3_pacbio_64_out.cnt.matrix.txt"

Read ReadCounts input files ->|
************************************************************ (0.00 sec)
Output results (0.00 sec)

Execute readCountsMatrix for VAF matrix...

readCountsMatrix Options:
ReadCounts Files (-c): cDN3_pacbio_64_out.txt
Matrix Output (-M): VAF
Min. Reads (-m): 5
Quiet (-q): False
Outfile File (-o): cDN3_pacbio_64_out.vaf-m5.matrix.txt

Command-Line: readCountsMatrix -c "cDN3_pacbio_64_out.txt" -M VAF -m 5 -o "cDN3_pacbio_64_out.vaf-m5.matrix.txt"

Read ReadCounts input files ->|
************************************************************ (0.00 sec)
Output results (0.00 sec)

[edwardsnj]

Hi MJ, I'm sorry you are still having trouble. I see that you didn't set a valid barcodes file on the command-line, in which case the default filename barcodes.tsv is used. You don't appear to have got an error due to this file being absent, so perhaps you made it (empty) to avoid the error, or it does not have barcodes that correspond to those in the BAM file (for some other reason). Note that the cell-barcode option selected CellRanger says that reads with cell-barcodes that are missing or not in the valid barcode file are dropped.

You can indicate that all cell-barcodes should be accepted using -b None to indicate there is no valid barcodes file, rather than leaving the option out, which says to use the default.

I suggest using "-b None" to see whether you get output without restricting the barcodes so you can see what scReadCounts finds. And try it with a small VCF file to start. :-) 18 hours is too long to wait for nothing.

[MJ-1987]

Hi,

I'll try that option but I have a barcodes.tsv file in the folder which has all the valid barcodes in it so it's not empty so I wouldn't think that that has caused the error. But I'll try with a small VCF file and -b None and see if it help.

[edwardsnj]

@MJ-1987 is SCReadCounts working for you now? I was hoping that if you turned off the valid barcodes option that you’d see whether CellRanger output the barcodes in the format you expected.

[MJ-1987]

Thanks for checking again. Yes, if I use the -b None parameter it work. thank you very much for your help.