Direct transposition of native DNA for sensitive multimodal single-molecule sequencing.

Area of Science:

• Genomics
• Molecular Biology
• Epigenetics

Background:

• Pacific Biosciences (PacBio) single-molecule sequencing of long DNA templates requires substantial input material.
• Current methods limit the application of PacBio sequencing for studies with limited DNA quantities.

Purpose of the Study:

• To develop novel tagmentation-based methods for PacBio sequencing that drastically reduce DNA input requirements.
• To enable multimodal single-molecule genomics, including genetic variation, DNA methylation, and chromatin accessibility analysis.

Main Methods:

• Adaptation of Tn5 transposition to fragment DNA and introduce hairpin oligonucleotides for generating PacBio-compatible circular molecules.
• Development of Single-Molecule Real-Time sequencing by Tagmentation (SMRT-Tag) for genetic variation and CpG methylation detection.
• Development of Single-Molecule Adenine-methylated Oligonucleosome Sequencing Assay by Tagmentation (SAMOSA-Tag) for chromatin accessibility profiling using exogenous adenine methylation.

Main Results:

• SMRT-Tag and SAMOSA-Tag utilize 90-99% less input DNA compared to existing protocols.
• SMRT-Tag with 40ng human DNA produced results comparable to gold standard whole-genome and bisulfite sequencing.
• SAMOSA-Tag resolved single-fiber chromatin structure, CTCF binding, and DNA methylation in prostate cancer xenografts, revealing epigenome disorganization associated with metastasis.

Conclusions:

• Tagmentation-based methods significantly lower DNA input requirements for PacBio single-molecule sequencing.
• These novel assays facilitate sensitive, scalable, and multimodal single-molecule genomics.
• The developed techniques hold promise for diverse basic research and clinical applications.