Systematic Botany, Justus Liebig University Gießen, Giessen, Germany.
PLoS One. 2021 Feb 2;16(2):e0245611. doi: 10.1371/journal.pone.0245611. eCollection 2021.
Pollen metabarcoding has received much attention recently for its potential to increase taxonomic resolution of the identifications of pollen grains necessary for various public health, ecological and environmental inquiry. However, methodologies implemented are widely varied across studies confounding comparisons and casting uncertainty on the reliability of results. In this study, we investigated part of the methodology, the effects of level of exine rupture and lysis incubation time, on the performance of DNA extraction and Illumina sequencing. We examined 15 species of plants from 12 families with pollen that varies in size, shape, and aperture number to evaluate effort necessary for exine rupture. Then created mock communities of 14 of the species from DNA extractions at 4 levels of exine rupture (0, 33, 67, and 100%) and two levels of increased lysis incubation time without exine rupture (2 or 24 hours). Quantities of these DNA extractions displayed a positive correlation between increased rupture and DNA yield, however increasing time of lysis incubation was associated with decreased DNA yield. Illumina sequencing was performed with these artificial community treatments with three common plant DNA barcode regions (rbcL, ITS1, ITS2) with two different primer pairings for ITS2 and rbcL. We found decreased performance in treatments with 0% or 100% exine rupture compared to 33% and 67% rupture, based on deviation from expected proportions and species retrieval, and increased lysis incubation was found to be detrimental to results.
花粉代谢条形码技术因其能够提高花粉粒鉴定的分类分辨率而受到广泛关注，这对于各种公共卫生、生态和环境研究是非常重要的。然而，由于研究中采用的方法多种多样，使得结果的可靠性存在不确定性，并且难以进行比较。在本研究中，我们研究了部分方法，即外壁破裂程度和裂解孵育时间对 DNA 提取和 Illumina 测序性能的影响。我们研究了来自 12 个科的 15 种植物的花粉，这些花粉在大小、形状和孔数上存在差异，以评估外壁破裂所需的努力程度。然后，我们从 14 种植物的 DNA 提取中创建了 14 种模拟群落，这些植物的外壁破裂程度分别为 0%、33%、67%和 100%，并且没有外壁破裂的情况下，裂解孵育时间分别为 2 小时和 24 小时。这些 DNA 提取的数量与增加的破裂和 DNA 产量呈正相关，但增加的裂解孵育时间与 DNA 产量的减少有关。我们使用这些人工群落处理方法进行了 Illumina 测序，其中包括三个常见的植物 DNA 条形码区域（rbcL、ITS1、ITS2），并使用两种不同的 ITS2 和 rbcL 引物对进行了配对。我们发现，与 33%和 67%的破裂相比，0%或 100%的破裂处理的性能下降，这是基于预期比例和物种检索的偏差，并且发现增加的裂解孵育时间对结果有害。
