Next-gen sequencing

Videos
http://www.youtube.com/watch?v=oYpllbI0qF8
http://gep.wustl.edu/curriculum/course_materials/gscmaterials_next.php

http://en.wikipedia.org/wiki/Shotgun_sequencing#Whole_genome_shotgun_sequencing

Coverage is the average number of reads representing a given nucleotide in the reconstructed sequence. It can be calculated from the length of the original genome (G), the number of reads(N), and the average read length(L) as NL / G. For example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2x redundancy.

next-gen sequencing has high coverage due to the high number of reads (but with shorter read lengths (anywhere from 25–500bp)) in a short amount of time and cheaper by parallelizing the sequencing process. assembly of these short reads is computationally expensive.

depth - number of sequence reads produced

http://en.wikipedia.org/wiki/Next-generation_sequencing#New_sequencing_methods

http://en.wikipedia.org/wiki/Pyrosequencing

Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle.

"Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The Pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemiluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are added and removed after the reaction, sequentially. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.


High-throughput sequencing

The high demand for low-cost sequencing has driven the development of high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once.[20][21] High-throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye-terminator methods.[22]
[edit] 454 pyrosequencing
Main article: 454 Life Sciences#Technology

A parallelized version of pyrosequencing was developed by 454 Life Sciences. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs.[16] This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.[23]
[edit] Solexa sequencing

Solexa has developed a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a slide and amplified so that local clonal colonies are formed (bridge amplification). One type of nucleotide at a time is then added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides and the dye is chemically removed from the DNA, allowing a next cycle.[24]
[edit] SOLiD sequencing
Main article: ABI Solid Sequencing

Applied Biosystems' SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide.[25] Similar to Solexa sequencing, this technology produces short read lengths at a low price per base.[23]

http://www.youtube.com/watch?v=nFfgWGFe0aA

amplicon sequencing - ultra deep, detect mutations at low level frequency in cancers
http://en.wikipedia.org/wiki/454_Life_Sciences#Amplicon_Sequencing

454 weakness:
A limitation of 454 sequencing remains resolution of homopolymer DNA segments; i.e. regions of template which contain multiple consecutive copies of a single base (A, C, G or T). Since pyrosequencing relies on the magnitude of light emitted to determine the number of repetitive bases, erroneous base calls can be a problem with homopolymers. Another disadvantage of 454 sequencing is that while it is cheaper and faster per base, each run is quite expensive, and it is therefore unsuited for sequencing targeted fragments from small numbers of DNA samples, such as for phylogenetic analysis