Our decade of experience in developing experimental and computational tools to study somatic mutations
has revealed temporal, cell-type specific, and mechanistic patterns of somatic mutations across multiple tissues
and across the human lifespan. Yet our view of somatic mutations has been limited due to cost. The field of
somatic mutations requires a technique with the flexibility of bulk tissue with the allele- and molecule-specific
power of single-cell techniques.
To build the unified somatic variants catalog of human genome the proposed research aims to develop
a Tn5 based duplex sequencing (Tn5-duplex-seq) method, a scalable single-well workflow to capture the
landscape of somatic mutations at the single-molecule level from a range of cell or DNA input using Tn5
transposase, which resolves complementary strands by strand orientation from simple PCR-based capture.
Duplex sequencing, the capture of complementary strand information in single molecules, offers unprecedented
accuracy for detection of somatic mutations, but can be error-prone or laborious depending on the method of
capturing complementary strands. The core concept of Tn5-duplex-seq has been successfully applied to single-
cells with high accuracy (META-CS) but requires optimization. In the proposed study we aim to 1) determine
parameters for pooled cell and bulk DNA sequencing to retain single-molecule information and cost-
effectiveness, and 2) harness the power of single-molecule duplex detection which provides the basis for a suite
of analytical tools to evaluate different types of somatic mutation including single nucleotide variants, indels,
microsatellites and copy number variants with high accuracy.
Tn5-duplex-seq can transform our understanding of somatic mutations through the innate
comprehensiveness of this technique: capturing both clonal as well as the rarest somatic mutations and providing
the basis of accuracy for analytical approaches to determine both somatic single base and structural variation.
Ultimately, our tool will be accessible, cost-effective, and scalable to be used by the larger scientific community
and easily integrated with pipelines of the Somatic Mosaicism across Human Tissues (SMaHT) Network. Tn5-
duplex-seq with comprehensive profiling of genetic mutations has the potential to answer fundamental questions
about our genomes in human biology and medicine.
This study will reveal the landscape of somatic mutations and their accumulation in human tissue at
single-molecule resolution, enabling detection of both clonal and cell-specific somatic mutations using
a flexible-input workflow. The proposed research is significant for the comprehensive, results-based
development of strategies for discovering the mutational burden and landscape in human development and
aging. Together with the planned characterization of mutational signatures, the anticipated results should
provide knowledge that may help to develop new strategies for preventing aging and disease progression.
