Abstract:
:High-throughput single-stranded DNA sequencing (ssDNA-seq) 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 at 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 DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. 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.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Wu DC,Lambowitz AMdoi
10.1038/s41598-017-09064-wsubject
Has Abstractpub_date
2017-08-21 00:00:00pages
8421issue
1issn
2045-2322pii
10.1038/s41598-017-09064-wjournal_volume
7pub_type
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