Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass--Sequence Relationships with an Innovative Metabarcoding Protocol.
Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass--Sequence Relationships with an Innovative Metabarcoding Protocol.
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DOI：
10.1371/journal.pone.0130324
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Leese F
中科院分区：
文献类型：
--
作者：
Elbrecht V;Leese F
元条形码是一种新兴的遗传工具，用于快速评估生态系统中的生物多样性。它涉及对来自环境样品的标准基因进行高通量测序，并与参考数据库进行比较。然而，在实验室筛选物种多样性和从环境样本中推断物种丰度的管道方面尚未达成共识。特别是，在批量处理样品时，尚未系统地检查低生物量标本的引物偏倚和检测限的影响。我们开发并测试了一种DNA元条形码协议，利用标准的细胞色素c氧化酶亚基I（COI）条形码片段来检测淡水大型无脊椎动物类群。大量提取DNA，在单个PCR步骤中扩增，并纯化，并在两个独立的MiSeq运行中直接测序文库（300-bp双端读段）。具体而言，我们评估了样本生物量对序列读段丰度的影响，通过对具有跨越生物量三个数量级的已知单倍型的石首鱼物种的31个样本进行测序（实验I）。然后，我们使用相同的标准条形码引物测试了52种不同的淡水无脊椎动物类群的相似生物量的回收率（实验II）。每个实验重复10次以最大化统计功效。两次重复实验的结果一致。我们发现物种生物量和MiSeq读数的数量之间存在明显的正相关性。此外，我们可靠地恢复了83%的52个类群用于测试引物的偏见。然而，序列丰度变化的四个数量级之间的分类群，尽管使用类似数量的生物量。我们的元条形码方法为高通量评估提供了可靠的结果。然而，结果表明，引物效率是高度物种特异性的，这将妨碍直接评估样品中的物种丰度和生物量。因此，基于PCR的生物多样性元条形码评估应该依赖于存在-不存在度量。
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.
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影响因子：
3.7
作者：
Hajibabaei M;Shokralla S;Zhou X;Singer GA;Baird DJ
通讯作者：
Baird DJ
DOI：
10.1098/rsbl.2003.0025
发表时间：
2003-08-07
影响因子：
4.7
作者：
Hebert, PDN;Ratnasingham, S;deWaard, JR
通讯作者：
deWaard, JR
使用DNA元法编码的环境状况评估：朝向基于遗传学的海洋生物指数（GAMBI）。
影响因子：
3.7
作者：
Aylagas E;Borja A;Rodríguez-Ezpeleta N
通讯作者：
Rodríguez-Ezpeleta N
影响因子：
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作者：
Haase, Peter;Pauls, Steffen U.;Sundermann, Andrea
通讯作者：
Sundermann, Andrea
影响因子：
7.7
作者：
Clarke, Laurence J.;Czechowski, Paul;Cooper, Alan
通讯作者：
Cooper, Alan