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Open Access Publications from the University of California

Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2016)

The advent of next generation sequencing (NGS) has fundamentally changed genetics research. Where researchers were once focused on sequencing the genome of a species, they now can sequence the genome of a particular tumor or even a single cell. NGS has also made it cost effective to sequence the RNA transcripts found within a cell, a collection of data known as the transcriptome. Unfortunately, current protocols for sequencing the genome and the transcriptome are incompatible. This leaves researchers with a choice: for a given sample you can examine either the DNA or the RNA. The work presented here makes it so that researchers no longer have to make this choice. This dissertation describes the development of a new protocol, known as Gel-Seq, that makes it possible to sequence both DNA and RNA from as few as 400 cells. This technology will allow researchers to directly examine the ways that changes in the genome impact the transcriptome. At the heart of the Gel-Seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a newly designed combination of polyacrylamide membranes that take advantage of the size differences between these molecules.

Two different device options were developed as a part of the Gel-Seq protocol. One device, designed for rapid adoption, was fabricated using standard equipment found inside of a biology laboratory. The second device, designed for separating low input samples, was fabricated using newly developed micro-scale fabrication techniques. In addition to the development of these physical devices, a biological protocol was developed to generate genome and transcriptome data using these devices. In order to validate this technology, a cell line with a stable genome and transcriptome was used. Comparing the Gel-Seq protocol to standard protocols, the results showed a high correlation for both the genome (R = 0.88) and transcriptome (R = 0.96) data. This supports the conclusion that the device can be used to produce correlated genome and transcriptome libraries. This dissertation reports on the development, optimization, and validation of the Gel-Seq protocol.

    Cover page: Gel-Seq: An Approach for Simultaneously Sequencing the Genome and Transcriptome in Small Populations of Cells
    • Article
    • Peer Reviewed
    Green Manufacturing and Sustainable Manufacturing Partnership (2013)
      Cover page: Quantifying the Improvements in Rapid Prototyping and Product Life Cycle Performance Created by Machining
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2018)
      The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.
        Cover page: Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2017)
        The advent of next generation sequencing has fundamentally changed genomics research. Unfortunately, standard protocols for sequencing the genome and the transcriptome are incompatible. This forces researchers to choose between examining either the DNA or the RNA for a particular sample. Here we describe a new device and method, collectively dubbed Gel-seq, that enables researchers to simultaneously sequence both DNA and RNA from the same sample. This technology makes it possible to directly examine the ways that changes in the genome impact the transcriptome in as few as 100 cells. The heart of the Gel-seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a newly designed device that contains several different polyacrylamide membranes. Here we report on the development and validation of this device. We present both the manufacturing protocol for the device and the biological protocol for preparing genetic libraries. Using cell lines with uniform expression (PC3 and Hela), we show that the libraries generated with Gel-seq are similar to those developed using standard methods for either RNA or DNA. Furthermore, we demonstrate the power of Gel-seq by generating a matched genome and transcriptome library from a sample of 100 cells collected from a mouse liver tumor.
          Cover page: Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers
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