PRJNA386569
Thermostable Group II intron reverse transcriptase single-stranded DNA-seq
High-throughput single-stranded DNA sequencing of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly from the 3' end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence E. coli genomic showed that the TGIRT enzyme has surprisingly robust DNA-dependent DNA polymerase activity for a reverse transcriptase. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends.