Cattonaro Federica, Spadotto Alessandro, Radovic Slobodanka, Marroni Fabio
IGA Technology Services Srl, Udine, Udine, 33100, Italy.
Department of Agricultural, Food, Environmental and Animal Sciences (DI4A), University of Udine, Udine, 33100, Italy.
F1000Res. 2018 Nov 8;7:1767. doi: 10.12688/f1000research.16804.4. eCollection 2018.
Shotgun metagenomics sequencing is a powerful tool for the characterization of complex biological matrices, enabling analysis of prokaryotic and eukaryotic organisms and viruses in a single experiment, with the possibility of reconstructing the whole metagenome or a set of genes of interest. One of the main factors limiting the use of shotgun metagenomics on wide scale projects is the high cost associated with the approach. We set out to determine if it is possible to use shallow shotgun metagenomics to characterize complex biological matrices while reducing costs. We used a staggered mock community to estimate the optimal threshold for species detection. We measured the variation of several summary statistics simulating a decrease in sequencing depth by randomly subsampling a number of reads. The main statistics that were compared are diversity estimates, species abundance, and ability of reconstructing the metagenome in terms of length and completeness. Our results show that diversity indices of complex prokaryotic, eukaryotic and viral communities can be accurately estimated with 500,000 reads or less, although particularly complex samples may require 1,000,000 reads. On the contrary, any task involving the reconstruction of the metagenome performed poorly, even with the largest simulated subsample (1,000,000 reads). The length of the reconstructed assembly was smaller than the length obtained with the full dataset, and the proportion of conserved genes that were identified in the meta-genome was drastically reduced compared to the full sample. Shallow shotgun metagenomics can be a useful tool to describe the structure of complex matrices, but it is not adequate to reconstruct-even partially-the metagenome.
鸟枪法宏基因组测序是表征复杂生物基质的有力工具，能够在单个实验中分析原核生物、真核生物和病毒，有可能重建整个宏基因组或一组感兴趣的基因。限制鸟枪法宏基因组在大规模项目中广泛应用的主要因素之一是该方法成本高昂。我们着手确定是否有可能使用浅度鸟枪法宏基因组来表征复杂生物基质，同时降低成本。我们使用了一个交错的模拟群落来估计物种检测的最佳阈值。我们通过随机抽取一定数量的 reads 来模拟测序深度的降低，测量了几个汇总统计量的变化。比较的主要统计量包括多样性估计、物种丰度以及在长度和完整性方面重建宏基因组的能力。我们的结果表明，对于复杂的原核生物、真核生物和病毒群落，500,000 条 reads 或更少就能准确估计多样性指数，不过特别复杂的样本可能需要 1,000,000 条 reads。相反，即使是最大的模拟子样本（1,000,000 条 reads），任何涉及宏基因组重建的任务表现都很差。重建组装的长度小于使用完整数据集获得的长度，与完整样本相比，在宏基因组中鉴定出的保守基因比例大幅降低。浅度鸟枪法宏基因组学可以是描述复杂基质结构的有用工具，但不足以重建（即使是部分）宏基因组。
