Sequencing saturation reflects the overall complexity and depth of sequencing for all fragments and can be obtained by calculating the redundancy of sequencing fragments with valid barcodes and UMIs that align to unique regions of the genome. The formula is: Sequencing Saturation = 1 - non-duplicated_unique_mapped_reads / total_unique_mapped_reads. For BAM files obtained through MobiVision Quantify, MAPQ=255 represents reads aligned to unique genome regions. Total_unique_mapped_reads can be obtained by calculating the number of sequencing fragments with corrected UMIs and barcodes in MAPQ=255 reads; non-duplicated_unique_mapped_reads can be obtained by counting non-duplicated sequencing fragments in MAPQ=255 reads with UMIs and barcodes. The code is as follows:

samtools view -q 255 Aligned.bam | gawk '{if (NF==16) {total_reads+=1; !umi[$19$20]++}} END {printf("%s%s\n" total_reads length(umi))}'