Sequencing Technology

There are now three main sequencing technologies that are available and commonly used: Illumina, PacBio and MinIon. Understanding the assumptions and limitations of each of these technologies can aid in planning the experimental design.

  • Illumina
    Illumina raw data are short (100-300bp) in size and of high quality for reads shorter than 200 bps. Quality scores for bases on reads between 250-300bp usually are of lower quality.
  • Size: short
  • Output: 90,000 Gb (HiSeq 3000-4000)
  • Base Quality: 99.9% High

  • PacBio PacBio raw data (subreads) are long (3,000-15,000bp) with max read lengths around 40,000-80,000bp.
  • Size: long
  • Output: 1 Gb (RSII)
  • Output: 1-5 Gb (Sequel)
  • Base Quality: 85% Medium

  • MinIon MinIon raw data are long (3,000-70,000bp) with max read lengths as high as 250,000bp.
  • Size: long
  • Output: 1 Gb (RSII)
  • Output: 1-5 Gb (Sequel)
  • Base Quality: 85% Medium
  • This paper provides a nice overview of MinIon sequencing technologies and uses Paper

Funding and Cost

Most research has a strict allowance for how much sequencing and bioinformatics can be performed to answer the biological question of interest. An understanding of the following terminology can aid in determining the type and amount of sequencing that is best suited for your biological purpose.

  • Read length:Short reads (50bp) are difficult to align to unique locations in a genome, so unless the experiment is for smRNA it is uncommon to use very short reads.

  • Paired-end Both ends of the DNA fragment are sequenced. This type of sequencing is useful for obtaining more unique alignments to a genome For RNA-Seq experiments with a known genome, it is recommended to use at least 100bp paired-end Illumina data. For RNA-Seq experiments without a genome or a genome of questionable quality, it recommended to use 150bp Illumina paired-end data.

  • Single-end Used when the experiment has DNA fragments shorter than the length of the read. For example, smRNA experiments are typically done with 50bp single-end data.

  • Biological Replicates It is extremely important to have at least 3 replicates and preferably 5 to 10 replicates for RNA-Seq experiments to determine differential expression


Examples

In the next sections we will go over several example experimental design problems from real world examples.

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