Fig. 1: NGS-based QASeq DNA absolute quantitation modules.

a Schematic of a single QASeq module for quantitation of DNA bearing a specific nucleotide sequence. Each input DNA strand was attached with a UMI by 2 PCR cycles. Further PCR amplification was performed after removing non-extended primers bearing UMI. NGS reads originating from the same input DNA strand carry the same UMI sequence, thus unique UMI family count represents the number of input DNA strands. b Quantitation of ERBB2 and EIF2C1 molecule count using 2-plex QASeq and ddPCR. Five replicated experiments were performed for both methods with 10 ng gDNA input. QASeq showed higher conversion yield (86% on average) than ddPCR (53% on average). The CV of molecule count was lower for QASeq (5.0% for ERBB2, 2.5% for EIF2C1) than for ddPCR (12.8% for ERBB2, 13.3% for EIF2C1). c Absolute quantitation of different DNA input. Observed ERBB2 molecule counts by QASeq were close to expected molecule counts calculated from DNA input amount because of high conversion yield. Lower conversion yield at 1 ng input DNA was possibly a result of material loss at low concentration. d Technical variation of ERBB2 ploidy using 2-plex QASeq. The mean of 5 replicates was 1.98, which is close to normal ploidy of 2. e UMI family size distribution and data processing. UMI family size follows log-normal distribution after removing small families. Family size cutoff was calculated as 5% of the mean of top 3 largest family size here.