It seems like only yesterday (okay, less than two years ago) that I learned about 454 sequencing. It’s the new technology that many folks think will replace dye termination Sanger sequencing using capillary arrays (the method used to sequence the human genome and many other genomes). A new technology is coming on the scene which may make 454 obsolete before it ever gets a foot-hold in the market (making it the laser disk of DNA sequencing).
454 sequencing works by copying small stretches of DNA sequence that have been attached to tiny beads. As each nucleotide is added to the growing sequence, light is emitted via a chemical reaction (this approach is known as pyrosequencing). The light is read by a sensor inside the machine and translated into a DNA sequence. Many sequencing reactions can be run in parallel in a single machine, generating millions of nucleotides of data within a couple of hours. The 454 technology is limited in that the individual reads are only 100-200 nucleotides long, which is short compared to Sanger reads that are approaching 1000 bases. Furthermore, pyrosequencing techniques struggle with mononucleotide repeats (stretches of DNA sequence of the same nucleotide, ie, AAAAA or CCCCC).
The newest, hippest, and sexiest sequencing technology comes from Solexa (see here). This approach allows one to generate 1 billion nucleotides of data in a single run (a few orders of magnitude more than 454). As with moving from Sanger to 454, the tradeoffs of moving from 454 to Solexa are mostly in read length. Solexa takes the sequencing in parallel strategy of 454 to a new level, but generates reads of only 25 nucleotides. As read lengths get shorter and shorter, assembly of the sequences into a complete genome becomes more difficult. But the sheer volume of sequence generated by the Solexa technology will force some people to take a closer look — it can sequence a complete human genome in a single run, albeit at 1x coverage.
The choice of which technology to use depends on the sequencing project one is carrying out. 454 has been shown to be useful for sequencing bacterial genomes de novo, but it’s not clear whether it will be practical for the large, more repeat rich genomes found in eukaryotes (although it seems to be a good strategy for sequencing ancient DNA). But short read lengths aren’t as big of a problem for resequencing projects — those that generate sequences from multiple individuals from a species with an already sequenced genome — because assembly of the reads can be done on the backbone of the previously published genome. The days of de novo genome sequencing with Solexa are far off, but I know of one group who plans to perform a large scale resequencing project using Solexa machines. This new technology may prove to be useful for genotyping individuals at disease associated loci or performing large scale population genetics surveys.