Chapter 1 Overview: Developments in DNA Sequencing

In 2002, the entire human genome was randomly sequenced within 6 month by Celera Genomics Corp with sequencing performed at an average rate of ∼9×109 bases/year with a factory of some 200 or more 96-capillary array electrophoresis sequencers. Recent improvements in the design of radial CAE microplate capillary channels and new matrix polymers can extend electrophoretic resolution and throughput, each by an order of magnitude or more above the sequencers used by Celera. Each 384-channel microchip sequencer can read ∼4 Mb per 24 hr. If an equivalent number of microchip sequencers were used today, their capacity would translate into 1.4×109 bases/year ×200=2.88×1011 bases/year, or about 10 human genomes sequenced to 10-fold coverage. Yet, this capacity is insufficient for planned levels of whole genome re-sequencing and de novo sequencing. Alternatively, new surface-array-based sequencing approaches that employ unitary base addition chemistries promise to make large-scale whole genome analysis a routine activity for smaller laboratories. Short-sequencing reactions over 30-120 nucleotides using new polymerase enzymes and sequencing chemistries such as pyrosequencing, cluster amplification and SBS on 'Clonal Single-Molecule Array', 'tSMS' and 'FISSEQ' methods, combine the enormous parallelism of microarrays with unitary base addition and with ultra-high-resolution optical systems for signal capture. Physical methods for large fragment sizing and sequencing using nanopore technologies and short fragments by fragmentation sequencing using mass spectrometry are also under rapid development of throughput, accuracy and sensitivity. The recent completion of a high-quality sequence of the human genome poses the challenge to understand the functional elements that it encodes, beyond the protein sequences that can be identified computationally. Comparative genomic analysis offers a powerful approach for finding such elements by identifying sequences that have been highly conserved during evolution. Margulies et al. (2005b) propose an initial strategy for detecting regions, which were highly conserved during evolution by generating low-redundancy sequence (∼2-fold redundancy) from a collection of 16 eutherian mammals, above the 7 mammals for which genome sequence data are already available in mid-2006. Margulies et al. (2006) also show that multi-sequence alignment methods are much better at aligning (and identifying) the available orthologous sequence from phylogenetically diverse vertebrates and contain significant amounts of both exonic and highly conserved non-exonic sequences that are the goal of such comparative sequencing programs. Although the data employed in these reports were from published sequence emanating from genomic Sanger sequencing programs, the potential to utilize sequence derived from SBS analysis for comparative alignment should be explored. The recent announcement of the planned sequencing of a complete Neanderthal genome (Homo sapiens neanderthalensis) by the 454 Life Sciences Corporations and the Max Planck Institute using SBS, underlines the importance of new sequencing technologies to post-genomic science. This project will be a major milestone for human genomic science. It represents the second human species to be sequenced, this time using extremely limited amounts of unique genetic material, where only an ultra-efficient technology could be conceived as making it possible. The scale of this project also represents a major stimulus for the development of the 454 Life Sciences Corporation's pyrosequencing technology and other short-read technologies. Attention to the development of specialist software and complementary technologies that utilize the data to the fullest will be other areas of rapid development. This will further stimulate interest from conventional life sciences in these platforms, as the ability to utilize the technologies and their data output is broadened. In that regard, another application of the pyrosequencing is for areas requiring the ability to sequence limited amounts of tumor material and for providing sufficient depth of coverage so as to identify rare genetic mutations occurring in a small proportion of the sample. By comparison the sensitivity of conventional Sanger CE sequencing of tumor biopsies is limited by the stromal contamination and by genetic heterogeneity within the cancer, and insufficient depth of coverage can be achieved economically to identify all mutations. Thomas et al. (2006) showed that GS20-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment, facilitating the accurate molecular diagnosis of cancer specimens within heterogeneous cellular genomes. New single molecule sequence approaches continue to be explored. Greenleaf and Block (2006) described a novel approach in which the sequence of DNA could be determined in principle by a motion-based method involving an ultra-stable optical trapping device capable of recording pauses in the motion of RNA polymerase when a limiting nucleotide limits the rate of transcription. Patterns of pauses could be aligned and a DNA sequence determined. See also: DNA sequencing, capillary array electrophoresis, silicon-chip microanalytical device, pyrosequencing, sequencing by synthesis (SBS), single molecule sequencing, sequencing by hybridization, tiling microarrays, sequencing by mass spectrometry, molecular pore sequencing, sequencing of ancient DNA, transcriptome sequencing

Valid recovery of nucleic acid sequence information from high contamination risk samples – Ancient DNA and environmental DNA

George A. Kowalchuk, Jeremy J. Austin, Paul S. Gooding and John R. Stephenhttp://www.elsevier.com/wps/find/bookdescription.cws_home/710463/description#descriptio