Next Generation Sequencing

In the new issue of Biotechniques, a paper by Maricic and Pääbo (login may be required) describes how a change to the standard 454 sequencing protocol  can dramatically increase the size of the library of DNA that goes into the actual sequencing reaction.

The trick used is to replace the last step in the library preparation where single stranded DNA is released from streptavidin beads. The original 454 protocol employes NaOH denaturation for this step, but the researchers found that this procedure results in a loss of over 99% of the DNA. However, When they replaced the NaOH denaturation with a heat denaturation by incubation recovery increased to 98%.

These authors are coming from the ancient DNA community and have an obvious motivation for optimizing the DNA retrieval from scarce ancient biological material. However, these findings are equally important to other applications aimed at sequencing small volumes of biological material such as tumors and within-host sub-populations of pathogens.