Big data: A method for obtaining large, phylogenomic data sets

January 9, 2014
The final annotated chloroplast genome assembly of Bartsia inaequalis with the 16 overlapping primer combinations indicated. Note that the primer combinations for regions 11, 12, 13, and 16 amplify both inverted repeat A and B in a single reaction. Credit: Photo by Simon Uribe-Convers from: Uribe-Convers, S., J. R. Duke, M. J. Moore, and D. C. Tank. 2014. A long PCR-based approach for DNA enrichment prior to next-generation sequencing for systematic studies. Applications in Plant Sciences 2(1): 1300063. doi:10.3732/apps.1300063

Traditional molecular systematic studies have progressed by sequencing genes one by one, a time- and cost-intensive task that has limited the amount of data a researcher could feasibly obtain. With the continual improvement of next-generation sequencing technologies, however, obtaining large molecular data sets is becoming much easier, and much cheaper. This increase in data means, in many cases, increased accuracy in reconstructing the evolutionary history of organisms.

As phylogenetic studies advance to include progressively more sequence data, new techniques are being developed to obtain such data sets. While it would be ideal to simply sequence entire genomes, this is not yet feasible across large numbers of taxa. Instead, current methods are being developed that allow researchers to target specific genomic regions of interest for the organisms being studied.

Scientists at the University of Idaho and Oberlin College have developed one such method to obtain large, phylogenomic data sets. "This method utilizes long PCR, or long-range PCR, to strategically generate DNA templates for next-generation sequencing," explains Simon Uribe-Convers, graduate student and lead author. The protocol is available for free viewing in the January issue of Applications in Plant Sciences.

Long-range PCR is a method that allows for the amplification of much larger fragments of DNA than is possible with traditional PCR—fragments larger than 40 kilobases have been reported in long PCR, versus fewer than 10 kilobases for traditional PCR. The authors of this study have developed a universal primer set across flowering plants that amplifies 3–15 kilobase fragments, which can then easily be sequenced using recently developed next-generation sequencing technologies. Uribe-Convers and colleagues tested this approach by amplifying chloroplast genomes for 30 species across flowering plants. Surprisingly, the primers were even found to successfully amplify chloroplast regions in several pine species. To further test the compatibility of this approach with next-generation sequencing, 15 complete chloroplast genomes (often referred to as plastomes) were then sequenced.

Although this study focused on plastomes and utilized the popular Illumina sequencing platform, Uribe-Convers explains, "[t]his can easily be expanded to mitochondrial and nuclear regions, and can be used in combination with any next-generation sequencing platform. Furthermore, this approach is not restricted to plant studies, but will be useful for any organism."

With the development of new methods such as the one described by Uribe-Convers and colleagues, scientists can obtain large, phylogenomic data sets for large numbers of taxa. Long-range PCR, in concert with next-generation sequencing, provides researchers with the means to sequence entire plastomes, mitochondrial genomes, and large portions of the nuclear genome.

"This method has important implications for the way future systematic studies are conducted as it provides researchers with a way to strategically target regions of interest in their study organism, such as single-copy regions of the nuclear genome or portions of organellar genomes, to produce large data sets at low costs," says Uribe-Convers. "We want to help move the field of systematics into the realm of big data, and we hope that our approach contributes to that."

Explore further: Analyzing complex plant genomes with the newest next-generation DNA sequencing techniques

More information: Simon Uribe-Convers, Justin R. Duke, Michael J. Moore, and David C. Tank. 2014. A long PCR-based approach for DNA enrichment prior to next-generation sequencing for systematic studies. Applications in Plant Sciences 2(1): 1300063. DOI: 10.3732/apps.1300063

Related Stories

Sequencing hundreds of chloroplast genomes now possible

January 31, 2013

Researchers at the University of Florida and Oberlin College have developed a sequencing method that will allow potentially hundreds of plant chloroplast genomes to be sequenced at once, facilitating studies of molecular ...

Sequencing without PCR reduces bias in measuring biodiversity

March 26, 2013

DNA barcode sequencing without the amplification of DNA by PCR beats the problem of false positives which can inflate estimates of biodiversity, finds a study published in BioMed Central and BGI Shenzhen's open access journal ...

Scientists re-imagine how genomes are assembled

November 25, 2013

Scientists at the University of Massachusetts Medical School (UMMS) have developed a new method for piecing together the short DNA reads produced by next-generation sequencing technologies that are the basis for building ...

A universal RNA extraction protocol for land plants

December 16, 2013

RNA, a nucleic acid involved in protein synthesis, is widely used in genetic research to study patterns of gene expression in different organisms. The types and quantities of RNA present in an organism indicate which genes ...

Recommended for you

Plastic in 99 percent of seabirds by 2050

August 31, 2015

Researchers from CSIRO and Imperial College London have assessed how widespread the threat of plastic is for the world's seabirds, including albatrosses, shearwaters and penguins, and found the majority of seabird species ...

Researchers unveil DNA-guided 3-D printing of human tissue

August 31, 2015

A UCSF-led team has developed a technique to build tiny models of human tissues, called organoids, more precisely than ever before using a process that turns human cells into a biological equivalent of LEGO bricks. These ...

Study shows female frogs susceptible to 'decoy effect'

August 28, 2015

(Phys.org)—A pair of researchers has found that female túngaras, frogs that live in parts of Mexico and Central and South America, appear to be susceptible to the "decoy effect." In their paper published in the journal ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.