Unable to recognize a combination of multiple barcode sequences

[shangyf-stu]

Hello,
I am processing data for library construction using BD Rhapsody and inDrop. Both of them contain combined barcodes. Barcode not found in *. BCstats.txt file

The barcodes in the file are null, but there are counts. I don't know what caused this error. Additionally, in the data of InDrop, there may be 1-2 mismatches in the W1 sequence (GAGTGATTGCTTTGTGACGCCTT) in the fastq file. How to set it in zUMIs?

To Reproduce
Yaml file:
project: dataset467
sequence_files:
file1:
name: /dataset467/fastq1/dataset467_1.fastq.gz
base_definition:
- cDNA(1-35)
file2:
name: /dataset467/fastq1/dataset467_2.fastq.gz
base_definition:
- barcode(1-8,31-38)
- UMI(39-44)
correct_frameshift: GAGTGATTGCTTGTGACGCCTT
reference:
STAR_index: /zUMIs_v2.9.7d/star_index_2.7.1a_nogtf
GTF_file: /3.2.0_star_index/Homo_sapiens.GRCh38.102.gtf
exon_extension: no
extension_length: 0
scaffold_length_min: 0
additional_files:
additional_STAR_params:
out_dir: /dataset467/zUMIs
num_threads: 8
mem_limit: 20
filter_cutoffs:
BC_filter:
num_bases: 1
phred: 20
UMI_filter:
num_bases: 1
phred: 20
barcodes:
barcode_num: null
barcode_file: null
barcode_sharing: null
automatic: yes
BarcodeBinning: 1
nReadsperCell: 100
demultiplex: no
counting_opts:
introns: yes
intronProb: no
downsampling: '0'
strand: 0
Ham_Dist: 0
velocyto: no
primaryHit: yes
multi_overlap: no
fraction_overlap: 0
twoPass: yes
make_stats: yes
which_Stage: Filtering
samtools_exec: samtools
Rscript_exec: Rscript
STAR_exec: STAR
pigz_exec: pigz

Screenshots
Error message:
Error in uik(bccount$cellindex, bccount$cs/1000) :
Method is not applicable for such a small vector. Please give at least a 5 numbers vector
Calls: cellBC -> .cellBarcode_unknown -> .FindBCcut -> uik
Execution halted
Error in fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, "kept_barcodes_binned.txt")) :
File '/dataset467/zUMIs/zUMIs_output/dataset467kept_barcodes_binned.txt' does not exist or is non-readable. getwd()=='/dataset467/zUMIs'
Execution halted
Loading required package: yaml
Loading required package: Matrix
Error in gzfile(file, "rb") : cannot open the connection
Calls: rds_to_loom -> readRDS -> gzfile
In addition: Warning message:
In gzfile(file, "rb") :
cannot open compressed file '/dataset467/zUMIs/zUMIs_output/expression/dataset467.dgecounts.rds', probable reason 'No such file or directory'
Execution halted
Error in data.table::fread(paste0(opt$out_dir, "/zUMIs_output/", opt$project, :
File '/dataset467/zUMIs/zUMIs_output/dataset467kept_barcodes.txt' does not exist or is non-readable. getwd()=='/dataset467/zUMIs'
Execution halted

I hope you can provide me with some help. Thank you!
Best wishes,
Shang

[shangyf-stu]

The yaml file above is for inDrop data, and the yaml file below is for BD Rhapsody:
project: dataset466
sequence_files:
file1:
name: /dataset466/filter_fastp/dataset466_clean_1.fastq.gz
base_definition:
- BD(1-9,22-30,44-52)
- UMI(53-60)
file2:
name: /dataset466/filter_fastp/dataset466_clean_2.fastq.gz
base_definition:
- cDNA(1-150)
reference:
STAR_index: /zUMIs_v2.9.7d/star_index_2.7.1a_nogtf
GTF_file: /3.2.0_star_index/Homo_sapiens.GRCh38.102.gtf
exon_extension: no
extension_length: 0
scaffold_length_min: 0
additional_files:
additional_STAR_params:
out_dir: /dataset466/zUMIs
num_threads: 8
mem_limit: 20
filter_cutoffs:
BC_filter:
num_bases: 1
phred: 20
UMI_filter:
num_bases: 1
phred: 20
barcodes:
barcode_num: null
barcode_file: null
barcode_sharing: null
automatic: yes
BarcodeBinning: 1
nReadsperCell: 100
demultiplex: no
counting_opts:
introns: yes
intronProb: no
downsampling: '0'
strand: 0
Ham_Dist: 0
velocyto: no
primaryHit: yes
multi_overlap: no
fraction_overlap: 0
twoPass: yes
make_stats: yes
which_Stage: Filtering
samtools_exec: samtools
Rscript_exec: Rscript
STAR_exec: STAR
pigz_exec: pigz

[cziegenhain]

Hi,

To your questions:

1. zUMIs does not support allowing for mismatches in the frameshift correction pattern
2. You do not receive useful output because your YAML files are incorrect. You must specify the barcode base ranges with BC but you have written barcode or BD. https://github.com/sdparekh/zUMIs/wiki/Protocol-specific-setup

Please check carefully all documentation before opening issues. Thank you.

[shangyf-stu]

Thank you for your answer. All samples have been successfully run. I'm sorry for the mistake caused by my carelessness!