Special News Feature – Sequencing Gems

Since the first description in 1977, DNA sequencing has held a fundamental place in molecular biology—enabling researchers to tease apart the basic building blocks of life and understand how organisms evolve and differ at the molecular level. It can also be argued that no single technology has evolved at a faster pace over the past three and a half decades, and no technology continues to advance at such a rapid pace with third- and even fourth-generation instruments in the works. With this in mind, the editors at BioTechniques have identified four articles from the pages of our journal that we considered worthwhile to highlight as Sequencing Gems for our 30 ^th anniversary. These articles join our previous Gem selections in PCR and biochemical methods. Presented in no particular order, each was important, or will be important, in DNA sequencing technology development.

Sensitive and Quantitative Universal Pyrosequencing Methylation Analysis of CpG Sites

2003

S. Colella, L. Shen, K.A. Baggerly, J.-P.J. Issa, and R. Krahe

Abstract: We developed a highly quantitative method to assess DNA methylation levels at specific sites using the Pyrosequencing technology (Pyrosequencing AB, Uppsala, Sweden). Pyrosequencing Methylation Analysis (PyroMethA) is a modification of the Combined Bisulfite Restriction Analysis (COBRA), where the restriction analysis is substituted with the highly quantitative Pyrosequencing reaction. Recently, Uhlmann et al. reported the independent development of a Pyrosequencing- based method to detect methylation. Our assay designed for a different gene further validates the use of the Pyrosequencing technology to detect methylation at specific CpG sites. In addition, we developed and validated universal PyroMethA as an approach for high-throughput methylation detection. Standard PyroMethA and universal PyroMethA

Epigenetic modifications, including DNA methylation, have been show to play essential roles in basic biological process as well as many human diseases. In fact, our growing knowledge of the widespread methylation within the genome and its effects on gene expression and inheritance has proved that genomes are much more complicated than scientists previously thought.

In this article by Colella et al., the authors demonstrate an assay to assess methylation states of CpG sites using pyrosequencing. The approach described in this article from 2003 is in many ways ahead of its time—today there exists a race among next-generation sequencing developers to create more robust methodologies for analysis of methylation status at a global level. While techniques such as bisulfite conversions might not be necessary in coming years as researchers develop new approaches to interrogate methylation without the need for such modifications, it is always interesting to recall where the such methods came from.

http://www.biotechniques.com/multimedia/archive/00011/03351md01_11443a.pdf

Fast, cost-effective development of species-specific microsatellite markers by genomic sequencing

2009

Jawad Abdelkrim, Bruce C. Robertson, Jo-Ann L. Stanton, and Neil J. Gemmell

Abstract: Microsatellites are the genetic markers of choice for many population genetic studies, but must be isolated de novo using recombinant approaches where prior genetic data are lacking. Here we utilized high-throughput genomic sequencing technology to produce millions of base pairs of short fragment reads, which were screened with bioinformatics toolsets to identify primers that amplify polymorphic microsatellite loci. Using this approach we isolated 13 polymorphic microsatellites for the blue duck (Hymenolaimus malacorhynchos), a species for which limited genetic data were available. Our genomic approach eliminates recombinant genetic steps, significantly reducing the time and cost requirements of marker development compared with traditional approaches. While this application of genomic sequencing may seem obvious to many, this study is, to the best of our knowledge, the first attempt to describe the use of genomic sequencing for the development of microsatellite markers in a non-model organism or indeed any organism.

At its core, sequencing is about diciphering single nucleotides and their arrangements in sequence. But over time, developers have discovered other applications for sequencing as well. This article demonstrated one of the growing uses of DNA sequencing outside traditional gene sequencing or whole genome sequencing. Here, and in other articles published recently, authors have demonstrated the ability to use next-generation sequencing platforms to identify polymorphic microsatellites for use in applications including evolutionary studies and forensic analysis.

http://www.biotechniques.com/multimedia/archive/00042/BTN_A_000113084_O_42484a.pdf

The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing.

1989

Del Sal, G., Manfioletti, G. and Schneider C.

Abstract: This report describes a common method of obtaining template DNA from phagemids, phages and plasmids. The strategy is based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) for DNA precipitation. By avoiding phase separation, many manipulation steps are reduced. A time-saving modification to perform double-stranded DNA sequencing directly after alkaline-denaturation is also introduced. The protocols described here allow the researcher to obtain template DNA from a variety of initial sources, thus giving reproducible sequencing results when using T7 DNA polymerase.

This article from Del Sal et al. in 1989 was an early effort to make sequencing easier and more accesible to researchers. The CTAB-DNA precipitation methodology presented a simple, useful, and efficient means to concentrate DNA samples prior to sequencing—enhancing throughput possibilities for applications such as direct sequencing of PCR products.

Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation

2012

Paul Coupland, Tamir Chandra, Mike Quail, Wolf Reik, and Harold Swedlow

Abstract: We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400–500 ng of sheared DNA for the library preparation.

Our final Gem article in the sequencing category is recent but could be a signal of the future. One of the major challenges for researchers working with next-generation sequencing platforms is the time and care needed in preparing sequencing libraries prior to placing reactions on an instrument. In this article by Coupland et al., the authors provide the first demonstration of the potential for direct sequencing without library preparation on a next-generation sequencing platform. While the researchers only used small-sized bacterial genomes, and much more work is required to enhance the output of the method, the first steps are in place and should lead to more improvements in the future.

http://www.biotechniques.com/multimedia/archive/00189/BTN5306-Coupland-1_189584a.pdf