lunes, 7 de marzo de 2016

Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol

Elbrecht V, Leese F (2015) Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol. PLOS ONE, 10, e0130324.


Seleccionado por: Nancy


Abstract

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It
involves high-throughput sequencing of a standard gene from an environmental sample
and comparison to a reference database. However, no consensus has emerged regarding
laboratory pipelines to screen species diversity and infer species abundances from environmental
samples. In particular, the effect of primer bias and the detection limit for specimens
with a low biomass has not been systematically examined, when processing samples in
bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome
c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate
taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the
libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end
reads). Specifically, we assessed the influence of specimen biomass on sequence read
abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning
three orders of magnitude in biomass (experiment I). Then, we tested the recovery of
52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding
primers (experiment II). Each experiment was replicated ten times to maximise statistical
power. The results of both experiments were consistent across replicates. We found
a distinct positive correlation between species biomass and resulting numbers of MiSeq
reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However,
sequence abundance varied by four orders of magnitudes between taxa despite the
use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for
high-throughput assessments. However, the results indicated that primer efficiency is highly
species-specific, which would prevent straightforward assessments of species abundance
and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity
should rely on presence-absence metrics.



Elegí este artículo porque me viene de perlas ahora que estoy en medio del establecimiento de un buen diseño de protocolo a seguir, para la parte experimental de mi proyecto de investigación. 1) Saber si la cantidad de ADN afecta en la diversidad en la comunidad de insectos. 2) Saber si realizar la extracción con nitrógeno líquido o TissueLyser afecta de alguna manera esta diversidad. Esto y otros puntos espero discutir con ustedes.

No hay comentarios:

Publicar un comentario