Banana Slug Genomics

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====== Pyrosequencing (454) ====== Wikipedia Page on 454 Sequencing: http://en.wikipedia.org/wiki/454_Life_Sciences //Text from a draft of Jenny Draper's Doctoral Thesis. **Please change.**// --- //[[learithe@soe.ucsc.edu|Jenny Draper]] 2010/03/30 16:30// ---- >Pyrosequencing, like most of the current high-throughput platforms, is an example of “sequencing by synthesis”, measuring the sequential incorporation of bases during the procession of DNA polymerase along the template. In pyrosequencing, the incorporation of each nucleotide is detected indirectly, by measuring the amount of pyrophosphate (PPi) released during the incorporation of the nucleotide into the growing DNA strand. A pyrophosphate detection assay is used to measure the degree of pyrophosphate release: an enzyme (ATP sulfurylase) converts the pyrophosphate into ATP; firefly luciferase then produces light in proportion to the quantity of ATP present. To avoid the necessity of washing between successive nucleotide additions, as required by previous sequencing-by-synthesis approaches, a nucleotide-degrading enzyme (apyrase) is included in the mix. This nucleotide-degrading enzyme is slow enough to allow the dNTP incorporation/PPi coversion/luciferase detection cycle to occur, but fast enough to prevent multiple incorporations of nucleotides, as well as thoroughly degrade all the dNTP and ATP present between each between each successive addition of nucleotides. > >Although pyrosequencing technology was conceived in 1987((Nyrén, P. The history of pyrosequencing. Methods Mol. Biol 373, 1-14 (2007).)) and in use by 1998((Ronaghi, M., Uhlen, M. & Nyren, P. DNA SEQUENCING: A Sequencing Method Based on Real-Time Pyrophosphate. Science 281, 363-365 (1998).)), it was not until 2005 that the process was implemented in a truly high-throughput instrument((Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376-380 (2005).)). Developed by 454 Life Technologies, this first “massively parallel” technology became known as “454 sequencing”. In 2008, a “barcoding” method was developed for the 454 system(( Meyer, M., Stenzel, U. & Hofreiter, M. Parallel tagged sequencing on the 454 platform. Nat. Protocols 3, 267-278 (2008).)), allowing sequencing of multiple samples on a single plate; all commercially available sequencing systems now have barcoding capability. > >To achieve high-throughput sample processing, millions of tiny beads, each containing only one sequence from the sample, are loaded onto a plate with millions of tiny wells, each large enough to contain only one DNA bead. Luciferase and sulfurylase are bound to “enzyme beads” which are likewise loaded into the wells; dNTPs are sequentially flowed over the entire plate, and an imager tracks light release from all wells on the plate simultaneously. For each dNTP, wells which light up have that nucleotide added to their sequence. > >Although each bead only has one DNA sequence bound, a single DNA molecule would be insufficient to generate the degree of light necessary for signal detection. Thus each bead needs many copies of the single sequence bound. This is achieved by “emulsion PCR”, in which each bead undergoes it’s own PCR reaction in a tiny droplet, resulting in each bead containing many copies of the original single sequence. These clonal populations of a single sequence are often referred to as a “polony”. > >Binding the DNA samples to the beads is achieved by ligating “adaptor” (or “linker”) sequences to the sample. The adaptors contain a 20bp region complementary to the PCR amplification primer each bead is coated with, providing the mechanism for attachment of the sample to the sequencing beads, and providing the primer necessary for the emulsion PCR amplification step. The adaptors also contain a region with a 20bp primer sequence for beginning the sequencing reaction, as well as a “barcode” sequence downstream of the sequencing primer, which is specific to each sample and can be used during sequence data analysis to assign the read to a specific sample group. > >The current 454 technology (the “GS FLX Titanium” series) can produce quality reads up to 500 bp in length – considerably longer than the other high-throughput methods currently available. However, the technology has two flaws. One is its lower read number (400-600 million reads and 1 gigabase of sequence per run)((GS FLX Titanium Series. @ http://454.com/products-solutions/system-features.asp#titanium)) compared to other technologies such as [[SOLiD]] (1.4-2.4 billion tags and 100-300 gigbases of sequence per run)((SOLiD™ 4 System. @ https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=607061&tab=DetailInfo)). The second is it’s difficulty in accurately detecting the length of runs of identical nucleotides – the “homopolymeric read problem”. This flaw, due to difficulty resolving the increased light signal when multiple identical dNTPs are integrated, produces a tendency to “call” an incorrect number of runs of identical nucleotides. This problem occurs with all four nucleotides, but is especially pronounced for adenine.