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Nathan Blow, Ph.D.
BioTechniques, Vol. 54, No. 1, January 2013, p. 14
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Adapter-less library construction

A key ingredient in today's massively parallel next-generation sequencing approaches is the creation of a sequencing library. Although several approaches have been advanced in recent years to construct these libraries they all generally require large amounts of starting material and additional purification steps such as gel extraction, placing restrictions on researchers. In this issue of BioTechniques, David Lazinksi and Andrew Camilli from Tufts University School of Medicine (Boston, MA) report on their development of a new technique for next-generation sequencing library preparation based on the use of homopolymer tails. Called homopolymer tail-mediated ligation PCR, or HTML-PCR for short, Lazinski and Camilli's approach starts with a controlled reaction to add homopolymer deoxycytosine (dC) tails to the 3’ ends of double stranded DNA. The addition of these tails enables the annealing and ligation of chimeric oligonucleotides with a user-defined sequence at the 5’ end and 4-7 complementary deoxyguanosines (dG). From here, the first annealed primer can be used in combination with a second primer possessing a long dG region complementary to the homopolymer dC tail and a second user-defined sequence in a PCR reaction to generate the final library products for sequencing. The authors tested their approach by creating Vibrio cholerae and Streptococcus pneumoniae libraries from nanogram and even sub-nanogram quantities of starting material, finding that HTML-PCR actually generated libraries that were at least partial effective down to 0.01ng of input material. In order to address the possibility that the homopolymer stretch of dG nucleotides in the second primer could amplify long oligo(dC) regions in the cases of larger genomes, the authors suggested the use of the artificial base 2-amino deoxyadenosine (2-amino dA) instead of dC for tailing and the use of deoxythymidines (dT) in the primers, since oligo(dT) primers can more stably anneal to 2-amino dA stretches than endogenous dA stretches in the genome. Unlike more the traditional approaches for sequencing library construction where adapters are directly ligated onto double stranded genomic DNA, HTML-PCR does not use adapters which enables the method to work with less starting material and eliminates the need for gel purification to remove adapter-dimers (gel purification can still be used for size selection if needed). And while other adapter-free methods have been published, HTML-PCR takes advantage of materials readily available in most molecular biology labs, making the technique accessible and cost-effective. In the end, these advantages also raise the interesting possibility that HTML-PCR could be further adapted in the future to work with high-throughput robotic applications.

See “Homopolymer tail mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction

Carrying the load

As any yeast biologist out there can attest, transforming yeast is a tricky business. This is due in large measure to the presence of a glycan- and mannose-rich cell wall acting as a barrier for the introduction of genetic material. Genetic transformation techniques, including lithium chloride-based chemical transformation, electroporation, and the use of gene gun technologies, have been described and refined. However, while specific approaches might prove useful with certain yeast species, there are several yeast species with strong potential for biotechnology applications where genetic transformation approaches would be highly desirable but the available methods have proven to be inefficient. Seeing the need for an improved transformation methodology, Filyak et al. detail the use an oligoelectrolyte polymeric carrier for yeast transformation in this issue of BioTechniques. The key to the author's new approach lies in the design of their polymeric carrier, which is composed of an anionic backbone with dimethyl aminoethyl methacrylate-based side branches. In direct comparisons with lithium chloride-based transformation and electroporation, the polymeric nanoscale carrier yielded at least two times more Hansenula polymorpha tranformants and five times more Pichia pastoris transformants. In addition, the polymeric carrier was as effective as lithium chloride and electroporation methodologies for the transformation of Saccharomyces cerevisiae, further confirming the wide range of yeast species this method can transform. The new polymeric carrier-based transformation approach described by Filyak et al. not only provides another tool for biologists in their quest to understand and genetically manipulate yeast species, but also presents a new avenue for exploration of genetic transformation technology in general as their technique is one of the first examples of the use of nanoscale carriers for DNA delivery in yeast.

See “A novel method for genetic transformation of yeast cells using oligoelectrolyte polymeric nanoscale carriers