SiNG-PCRseq: Accurate inter-sequence quantification achieved by spiking-in a
neighbor genome for competitive PCR amplicon sequencing
Soo A Oh1, Inchul Yang2, Yoonsoo Hahn3, Yong-Kook Kang4, Sun-Ku Chung1
and Sangkyun Jeong1,*
1. Medical Research Division, KIOM, Daejon 305-811, Koera
2. Center for Bioanalysis, KRISS, Daejon 305-340, Korea
3. Department of Life Science, Chung-Ang University, Seoul 156-756, Korea
4. Development and Differentiation Research Center, KRIBB, Daejon 305-806, Korea
SiNG-PCRseq compares the DNA sequences quantitatively to matched sequences in a
neighbor genome to reveal an unbiased inter-sequence quantitative landscape. This method
took advantage of genome-wide chemically equivalent, but discriminable homologous
sequences with a known copy arrangement in the neighbor genome. We tested this concept
using NGS-assisted quantitative competitive PCR for human samples with a spike-in of
orangutan genomic DNA (gDNA). Analysis of human gDNA validated SiNG-PCRseq in
accurately representing the quantitative relationships of many different sequences in the
genome (RMSD = 0.03 and 0.05 in two separate measurements (n = 367 and 299,
respectively)). Quantitation of cDNA sequences also resulted in excellent correlations
between technical replications (R2 = 0.93 and 0.92 for two human cell lines), proving the
consistency and reproducibility of our technique. Given the results insofar as being obtained
and the measurement structure explicitly relating the quantities of different sequences, SiNGPCRseq substantiates its utility in a range of applications. For instance, the SiNG-PCRseqyielded quantitative landscape for those cDNA sequences appeared relatively discordant to
RNA-seq-derived version in inter-sequence quantitation (R2 = 0.44 and 0.46) but highly
concordant in inter-sample quantitative comparison (R2 = 0.88), providing an insight into the
sequence dependent quantitative biases in RNA-seq. Not only that, SiNG-PCRseq via its
simple and relatively high-throughput analytic capability could well suit in determining copy
number differences, relative quantitating the transcript variants and inter-species quantitative
comparison of gene expressions.