Next-Generation-Sequencing? Try THIS-Generation-Sequencing. (A great talk about NGS by Elaine Mardis here: https://www.youtube.com/watch?v=6Is3W7JkFp8)
The basic principles of DNA sequencing.
Chemical Sequencing (Maxam-Gilbert)
Chain termination (Sanger)
Shotgun Sequencing - Introduction to the problem of assembly
Pause: It's all about the chemistry. Look at the reaction. (Fluoresence, ATP, H+). Remember: Chemistry is never 100%
Pyrosequencing. Combination of four enzyme to monitor luminesence produced by the ATP produced in series of bound nucleotides. (454-Roche) (https://www.youtube.com/watch?v=nFfgWGFe0aA)
Sequence by synthesis. Elongation of a template by serial addition of fluorescent-labeled nucleotides. Illumina (https://www.youtube.com/watch?feature=player_embedded&v=HMyCqWhwB8E)
Sequencing by ligation. All possible fluorescent-labeled di-nucleotides are catalyzed to bind by a DNA-ligase due to their differential affinity. 2-rounds of ligation (each nucleotide is read twice). SOLiD
Single-molecule-real-time (SMRT) Sequencing. A single DNA polymerase attached to a single molecule with a zero-mode-waveguide (ZMW). Fluorescent labeling for each nucleotide. PacBio. (http://www.sciencemag.org/content/323/5910/133.abstract) (https://www.youtube.com/watch?v=NHCJ8PtYCFc)
Ion Semiconductor Sequencing. Sequence by synthesis without fluorescent-labels. Synthesis is monitored through micro-changes in the pH by the creation of protons via the ligation process. (Ion Torrent, Ion Proton)
Various Platforms
454 Roche.
Illumina.
SOLiD.
SMRT.
Ion Proton.
Table with specifications of read lengths, throughput, price etc.
Steps before
Isolate DNA/RNA
Shear/Fragment
Partition of Genome (Hybrid Capture: create biotynilated probes for every fragment of interest)
Library construction
Amplification
Immobilization
Sequencing Costs through the years (Source: NHRI) http://en.wikipedia.org/wiki/File:Historic_cost_of_sequencing_a_human_genome.svg
Terminology
Throughput
Read
Reference Genome
Barcoding, Indexing
Coverage (of genome)
Depth (per nucleotide)
Applications of NGS
Genome Sequencing (DNA Sequencing, Resequencing of Genomes)
Gene Expression Analysis (RNASeq, Sequencing of cDNA)
Analysis of DNA binding protein localization (ChIPSeq, Sequencing coupled with ChIP, but also MNaseSeq and others)
Analysis of chromatin structure (DNaseSeq, FAIRESeq)
Genomic Variation (Deep Sequencing of exons, WES or total DNA)
Genome Structure (3C-4C-5C, HiCSeq, Sequencing of segments enriched for chromosomal contacts, ChIAPET)
In general. ANY form of enrichment of DNA fragments can be coupled to a NGS approach
Once you get the data:
Quality control
Mapping
Various downstream analysis depending on the experiment