1. Background
  2. Data Collection Methods
  3. Data Quality Control
  4. Summary

Illumina NextSeqTM 500 System. Image source: Product brochure (2014).

Gene Expression Data

  1. Methods to obtain data about gene expression on a small scale include:
    1. qPCR
    2. Serial Analysis of Gene Expression (SAGE)
  2. For transcriptomics, two approaches dominate data generation (Wang et al., 2009):
    1. DNA microarrays - commercially introduced in 1996 (Friend and Stoughton, 2002)
      1. Requires gene probes (single-strands of DNA) attached to an array slide
      2. Hybridization of fluorescently labelled cDNA reverse transcribed from sampled mRNA emits a measureable fluorescent signal
      3. Advantages: relatively inexpensive
      4. Disadvantages: requires knowledge about the genome sequence, limited sensitivity (high background levels) and dynamic range.
    2. Since 2006, RNA sequencing (RNA-seq) has gained popularity over DNA microarrays for transcriptomics. (McGettigan, 2013).
      1. Advantages include: direct determination of cDNA sequences, and a large dynamic range.
      2. Disadvantages include: Current expense, and extremely large data files (on the order of terabytes).

      The remainder of this module focuses on the RNA-Seq method.