1. Bioinformatics Lectures at Rice
Lecture 2: High throughput technologies in genomics
By Li Zhang

2. Microarrays Biology: The biological problems
Technology: Microarray mechanism; experimental procedures
Statistical methods: data analysis, checking quality, exploration, discovery.

3. Microarray technology
Microarray technology measure copy number of molecules in a mixture on a small slide.
Thousands or millions of different kinds of molecules can be measured simultaneously, thus creating large volumes of data per biological sample.
The molecules can be DNA, RNA or protein.

4. Major types of microarrays
Two color short oligo arrays
Single color short oligo arrays
Synthesized by photolithography:
(Eric Lander)
Bead arrays

5. The experimental procedure to produce microarray data
Affymetrix Gene expression Analysis Sample preparation protocol:
RNA isolation
cDNA synthesis
cRNA synthesis
Hybrdization
Amplification
Scan

6. Targets of Microarray measurements
mRNA gene expression
SNP genotyping
DNA copy number (aneuploidy, chromosomal aberration,LOH)
DNA methylation
ChIP-chip. Protein-DNA binding site
Nucleosome binding site

7. Some key aspects of microarray technology
Parellel. The technology is design to measure a larger number of different molecules.
Almost comprehensive. It can work for some or most of the molecules, but not for all, which will result in some missing data.
Noise and bias. The signals can be affected by unwanted source, e.g., cross-hybridization, which creates biases. Contamination also may have asymmetrical distribution.
Nonlinear response. Saturation causes non-linear behavior.
Evolving annotation. Identity of the molecules may change, reflecting new knowledge through time.
No units. The numbers are often on relative scale, which means the data have are not been calibrated.

8. Next generation sequencing techniques

9. Sequence by synthesis on an array
Illumina/SOLiD/454 Life sciences
(1.5 hr video, from a meeting in 2010)
Illumina’s animation. ( (3 min)
Solid’s animation.
Complete Genomics ( Nanoball sequencing).

10. Nano-ball of Complete Genomics

11. Some key aspects of next generation sequencing technology
Compared with microarrays, NGS has less noise, no cross hybridization, and no saturation.
Bias remains a problem. Some sequences simply cannot be dealt with properly. These include high GC sequences, repeats, etc.
Mapping to the genome can be challenging. But paired-ends help a lot.
Biases partly come from PCR amplification, whose efficiency differ depending on the sequences.

12. 3rd Generation sequencing
Single molecule, with no PCR amplification.
No fluorescence dyes, hence less reagent cost.
Longer sequences
Remaining problem: erratic base calling.
Ion torrent (
Pacific Biosciences (
Nano-pores (

13. Challenges ahead Complexity of human diseases Heterogeneity
Biological samples are fragile, subject to degradation, contamination.
Biases, batch effects, standards.