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Discrepancy in plasmid and transposons copy number estimation using WGS vs qPCR
We are trying to estimate plasmid copy number in a bacterium by mapping Illumina reads on corrected PacBio contigs and comparing read coverage between the chromosomal and plasmid contigs. Chromosome is about 5 Mbp and plasmid is 190 kbp.
The depth was good for both contigs (200-1000 reads). The ratio between chromosomal vs plasmid was about 1:2. The coverage of a transposon in the plasmid was even higher (1:4 comparing to chromosomal). I tried to confirm this using qPCR by comparing PCR efficiency-adjusted Ct (not the typical ddCT that assumes all efficiency = 2) between two reference single-copy chromosomal genes (rpoB, gyrA) and three target genes on the plasmid (two inside a transposon and one outside), and everything came down to a tight and solid 1:1. It is not uncommon for a plasmid like this to be single copy. I was wondering if the sequencing depth could be used in this manner without any bias adjustment. I am seriously doubting my ability to design a PCR, but I found nothing wrong with it. Should I trust the simple coverage calculation or the PCR at this point?
The average GC content of chromosome is about 56%. Plasmid GC content is 54%, but the transposon on the plasmid (10 kb) is 62% GC.
Any suggestion would be highly appreciated. Thank you so much!
The depth was good for both contigs (200-1000 reads). The ratio between chromosomal vs plasmid was about 1:2. The coverage of a transposon in the plasmid was even higher (1:4 comparing to chromosomal). I tried to confirm this using qPCR by comparing PCR efficiency-adjusted Ct (not the typical ddCT that assumes all efficiency = 2) between two reference single-copy chromosomal genes (rpoB, gyrA) and three target genes on the plasmid (two inside a transposon and one outside), and everything came down to a tight and solid 1:1. It is not uncommon for a plasmid like this to be single copy. I was wondering if the sequencing depth could be used in this manner without any bias adjustment. I am seriously doubting my ability to design a PCR, but I found nothing wrong with it. Should I trust the simple coverage calculation or the PCR at this point?
The average GC content of chromosome is about 56%. Plasmid GC content is 54%, but the transposon on the plasmid (10 kb) is 62% GC.
Any suggestion would be highly appreciated. Thank you so much!