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Bethyl Laboratories
Bethyl Laboratories

Detecting Diversity

High-throughput sequencing without PCR could make estimates of biodiversity more precise.

By | November 1, 2013

© GEORGE RETSECKIn the pregenomic era, biodiversity monitoring generally involved collecting species and identifying them by sight—a process fraught with human error. Once DNA sequencing became commonplace, however, this practice was largely replaced by DNA barcoding, which identifies species through their unique sequence differences at a particular gene. For instance, the most common barcoding gene used for identifying animals is mitochondrial cytochrome c oxidase subunit 1 (CO1).

Today, high-throughput next-generation sequencing enables the simultaneous barcoding of a mix of species, so that, for example, all insects collected in a trap can be processed at once. However, this approach requires PCR amplification of CO1, which introduces the potential for certain species to be over- or underrepresented when primers—small pieces of single-strand DNA that initiate PCR amplification—are better matched to one species’ DNA sequences than to another’s.

To avoid such bias, Xin Zhou and his colleagues at the research institution BGI in Shenzhen, China developed an approach that avoids PCR altogether. Instead, his team used mitochondrial enrichment to increase the proportion of CO1 DNA. Seventy-three insects from BGI’s local habitat were thrown in a blender, and the resulting soup was centrifuged at speeds that enabled isolation of a mitochondria-enriched fraction. The team then performed ultra-high-volume sequencing to recover CO1 DNA and identify the insect species present.

Even the new approach is not trouble-free, however. “It’s a good proof of concept,” says Mehrdad Hajibabaei, a professor of integrative biology at the University of Guelph in Ontario, Canada. But the increased sequencing volume necessary to compensate for the lack of PCR increases the cost, he says. (GigaScience, 2:4, 2013; published by BGI)

MONITORING METHOD
 
SEQUENCING PLATFORM COST TO ANALYZE A BATCH OF INSECTS SPECIES IDENTIFIED DETECTS RELEVANT ABUNDANCE
PCR amplification of CO1 genes followed by high-throughput sequencing 454 Genome Sequencer FLX + System ~ $100 ~ 90% No
Mitochondrial enrichment followed by high-throughput sequencing Illumina HiSeq 2000 This small-scale, proof of concept sample cost $600, but larger-scale samples may cost more. 92–97% Yes

 

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