PROJECT SUMMARY/ABSTRACT:
Single cell sequencing technologies have rapidly advanced over the past five years and have become
essential tools in a broad array of fields, ranging from developmental biology to human genetics. One major
advance was the development of high-throughput barcoding of RNA or DNA within droplets, which allowed for
preparation of sequencing libraries as a pool rather than in a laborious 96 well plate format. This advance
enabled sequencing analysis of thousands of cells in one batch. However, there has not been the same
breakthrough in scale when sequencing both DNA and RNA from the same cell as established techniques
involve sorting cells into plates or wells and ultimately separating DNA and RNA before library preparation. The
separation of RNA from DNA is necessary because conventional PCR amplification requires a 95oC melting
step, which degrades RNA. In this proposal, we will seek to optimize DNA amplification through recombination
polymerase amplification (RPA), an isothermal form of PCR which can be performed simultaneously with
reverse transcription of mRNA into cDNA. In this way, both RNA and DNA can be captured into barcoded,
sequencing libraries within the same droplet. The technology could have impact in many different fields in
which genotype-phenotype correlation is needed, such as characterizing genetic polymorphisms or genetic
variants in cancer. In SA1, we will demonstrate proof-of-principle of same cell DNA amplicon and RNA
transcriptome technology via isothermal RPA and reverse transcription. In SA2, we will determine the
quantitative performance of the methodology in two use applications and test capability to genotype
heterozygous alleles. If successful, this methodology could be widely adopted by laboratories in diverse fields
to answer questions related to genotype-phenotype correlations.
