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 GigaScience. This method tested on a bulk 'squashome' of mixed insect samples is also able rapidly and cost-effectively estimate biomass.

Often samples collected in the field are too small be sequenced directly. Traditionally, to get round this problem, DNA from the sample is amplified using multiple cycles of PCR. However this process is highly dependent on the chemical and physical properties of each piece of DNA and consequently this approach can selectively amplify certain sequences more than others.

Researchers from BGI and the China National -Shenzhen have developed a method of COI metabarcoding which uses mitochondrial enrichment prior to , rather than PCR, to ensure a high enough concentration of DNA for next generation ultra-deep sequencing to be accurate.

Successfully tested on 69 collected from a mountain in sub-tropical China this method was able to correctly match insects to their reference samples without introducing the false positives often seen using traditional methods. False positives are the main culprit behind biodiversity inflation. A few samples from insects with a body length of less than 5mm contained too little material for this method to work.

The screen also managed to identify bacteria within the samples, two of which were Wolbachia, a frequently found in insects, plus Legionellaceae and Bartonellaceae which can cause disease in humans. Additionally DNA was found from a member of the Lepidoptera order (butterflies and moths) even though it was not in the original insect mixture. It is thought that this probably either came from the gut of an insect that ate it, or from an undetected fragment or egg.

Commenting on the ability of this study in evaluating biodiversity Dr Xin Zhou who led this study commented, "We also found that sequencing 'volume' (nucleotide numbers) strongly indicated total biomass for the species in a bulk sample. By being able to identify both species and the biomass for each species more accurately than traditional PCR-based sequencing, this method could revolutionise biodiversity research and biomonitoring."

Explore further: Bug splatter on your car's windshield is a treasure trove of genomic biodiversity

More information: Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification, Xin Zhou, Yiyuan Li, Shanlin Liu, Qing Yang, Xu Su, Lili Zhou, Min Tang, Ribei Fu, Jiguang Li and Quanfei Huang GigaScience 2013, 2:4 doi:10.1186/2047-217X-2-4

NGS biodiversity data
Zhou, X; Li, Y; Liu, S; Yang, Q; Su, X; Zhou, L; Tang, M; Fu, R; Li, J GigaScience Database 2013

NGS Biodiversity software
Zhou, X; Li, Y; Liu, S; Yang, Q; Su, X; Zhou, L; Tang, M; Fu, R; Li, J; Huang, Q, GigaScience Database 2013 (Provides software and supporting material)

Related Stories

New method for sequencing genome in a single cell

December 21, 2012

(—The traditional genome sequencing process requires thousands of cells (or more) to provide sufficient DNA, and this means that variations that are only present in a small number of cells―such as early cancer ...

Improving DNA amplification from problematic plants

January 3, 2013

The polymerase chain reaction (PCR) is a common technique used to amplify, or copy, pieces of DNA. Amplified DNA is then used in genetic analyses for everything from medicine to forensics. In plant research, PCR is a vital ...

Recommended for you

Genomes uncover life's early history

August 24, 2015

A University of Manchester scientist is part of a team which has carried out one of the biggest ever analyses of genomes on life of all forms.

Rare nautilus sighted for the first time in three decades

August 25, 2015

In early August, biologist Peter Ward returned from the South Pacific with news that he encountered an old friend, one he hadn't seen in over three decades. The University of Washington professor had seen what he considers ...

Why a mutant rice called Big Grain1 yields such big grains

August 24, 2015

(—Rice is one of the most important staple crops grown by humans—very possibly the most important in history. With 4.3 billion inhabitants, Asia is home to 60 percent of the world's population, so it's unsurprising ...


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.