Issue with Analysis - atac_pbmc_5k_nextgem_scATAC_validcells.txt File is Empty

[AfterDream]

During the analysis, two issues were encountered:

An error code was displayed during the analysis bedtools slop -i Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_all_treat_pileup.bdg -g /home/bioinfo/miniconda3/envs/MAESTRO/lib/python3.8/site-packages/MAESTRO/annotations/GRCh38_chrom_len.txt -b 0 | /home/bioinfo/miniconda3/envs/MAESTRO/lib/python3.8/site-packages/MAESTRO/utils/bedClip stdin /home/bioinfo/miniconda3/envs/MAESTRO/lib/python3.8/site-packages/MAESTRO/annotations/GRCh38_chrom_len.txt Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.clip;sort -k1,1 -k2,2n Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.clip > Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.bdg;/home/bioinfo/miniconda3/envs/MAESTRO/lib/python3.8/site-packages/MAESTRO/utils/bedGraphToBigWig Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.bdg /home/bioinfo/miniconda3/envs/MAESTRO/lib/python3.8/site-packages/MAESTRO/annotations/GRCh38_chrom_len.txt Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem.bw;rm Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.bdg;rm Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.clip Then the error message reads as follows: [Error - overlapping regions in bedGraph line 242572 of Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_sort.bdg]. I resolved this by using awk to remove duplicate rows in the third column, after which the process ran successfully.

However, when running the following command: [MAESTRO scatac-peakcount --binary --peak Result/Analysis/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_final_peaks.bed --fragment Result/Mapping/atac_pbmc_5k_nextgem/fragments_corrected_dedup_count.tsv --barcode Result/QC/atac_pbmc_5k_nextgem/atac_pbmc_5k_nextgem_scATAC_validcells.txt --species GRCh38 --cores 4 --directory Result/Analysis/atac_pbmc_5k_nextgem --outprefix atac_pbmc_5k_nextgem], it was discovered that the atac_pbmc_5k_nextgem_scATAC_validcells.txt file is empty. This has resulted in the subsequent analysis being unable to proceed.

My software version is 1.5.1, the Python version is 3.8.0, and the dataset used is from the download link in the software documentation. Could you please help me solve this problem?