I recently sequenced some bisulfite converted DNA and some unconverted DNA in the same lane of a MiSeq. In the bisulfite converted DNA I had spiked in a small amount of unmethylated lambda DNA as a control. In the unconverted DNA I did not spike in the lambda control. I mapped both the unconverted and converted DNA to the lambda genome with stringent mapping parameters using Bismark. Both samples gave me alignments. It appears that the unconverted sample mapped only to the unconverted lambda genome where as the converted samples mapped to converted lambda genomes. However, how can I confidently determine my conversion efficiency if the results could be due to nonspecific mapping? Has anyone had this problem?
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NGS has revolutionized clinical microbiology by offering new ways to identify and surveil pathogens without prior knowledge of the target. One popular approach is 16S rRNA and ITS amplicon sequencing, which targets specific microbial markers. But studies are increasingly incorporating shotgun metagenomics, a technique that sequences all DNA from a sample without targeting specific microbial markers. This approach allows more detailed taxonomic classification,...10-30-2026, 06:46 AM