Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding.
Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding.
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DOI：
10.1038/s41598-017-17333-x
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发表时间：
2017-12-15
影响因子：
4.6
通讯作者：
Gillespie RG
中科院分区：
文献类型：
--
作者：
Krehenwinkel H;Wolf M;Lim JY;Rominger AJ;Simison WB;Gillespie RG
基于扩增子的元条形码有望快速、经济地分析物种组成。然而，它是有争议的丰度估计是否可以来自元条码由于分类群特异性PCR扩增的偏见。已经提出了无PCR的方法来缓解这个问题，但是伴随着工作量和成本的显著增加。在这里，我们分析了不同节肢动物群落的多位点数据集，以评估扩增偏倚是否可以通过（1）用高度简并引物或保守的引发位点靶向位点，（2）增加PCR模板浓度，（3）减少PCR循环次数或（4）通过直接测序基因组DNA避免位点特异性扩增来抵消。通过简并引物或具有保守引发位点的靶向扩增子，扩增偏倚显著降低。令人惊讶的是，PCR循环的减少对扩增偏倚没有强烈的影响。分类单元丰度和读段计数的关联实际上在更少的周期中不太可预测。即使完全排除位点特异性扩增也不能排除偏倚。靶基因座的拷贝数变异可能是分类群之间读段丰度差异的另一种解释，这将同样影响基于扩增子的方法和无PCR的方法。由于读丰度偏差是分类群特定的和可预测的，校正因子的应用允许丰度估计。
Amplicon based metabarcoding promises rapid and cost-efficient analyses of species composition. However, it is disputed whether abundance estimates can be derived from metabarcoding due to taxon specific PCR amplification biases. PCR-free approaches have been suggested to mitigate this problem, but come with considerable increases in workload and cost. Here, we analyze multilocus datasets of diverse arthropod communities, to evaluate whether amplification bias can be countered by (1) targeting loci with highly degenerate primers or conserved priming sites, (2) increasing PCR template concentration, (3) reducing PCR cycle number or (4) avoiding locus specific amplification by directly sequencing genomic DNA. Amplification bias is reduced considerably by degenerate primers or targeting amplicons with conserved priming sites. Surprisingly, a reduction of PCR cycles did not have a strong effect on amplification bias. The association of taxon abundance and read count was actually less predictable with fewer cycles. Even a complete exclusion of locus specific amplification did not exclude bias. Copy number variation of the target loci may be another explanation for read abundance differences between taxa, which would affect amplicon based and PCR free methods alike. As read abundance biases are taxon specific and predictable, the application of correction factors allows abundance estimates.
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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
影响因子：
16.6
作者：
Fonseca, Vera G.;Carvalho, Gary R.;Sung, Way;Johnson, Harriet F.;Power, Deborah M.;Neill, Simon P.;Packer, Margaret;Blaxter, Mark L.;Lambshead, P. John D.;Thomas, W. Kelley;Creer, Simon
通讯作者：
Creer, Simon
影响因子：
15.5
作者：
Angly FE;Dennis PG;Skarshewski A;Vanwonterghem I;Hugenholtz P;Tyson GW
通讯作者：
Tyson GW
影响因子：
3.7
作者：
Elbrecht V;Leese F
通讯作者：
Leese F
影响因子：
9.2
作者：
Drummond AJ;Newcomb RD;Buckley TR;Xie D;Dopheide A;Potter BC;Heled J;Ross HA;Tooman L;Grosser S;Park D;Demetras NJ;Stevens MI;Russell JC;Anderson SH;Carter A;Nelson N
通讯作者：
Nelson N