Marynowska Martyna, Goux Xavier, Sillam-Dussès David, Rouland-Lefèvre Corinne, Roisin Yves, Delfosse Philippe, Calusinska Magdalena
Luxembourg Institute of Science and Technology, 41 rue du Brill, L-4422, Belvaux, Luxembourg.
Institute of Research for Development - Sorbonne Universités, Institute of Ecology and Environmental Sciences - Paris, U242, 32 avenue Henri Varagnat, F-93140, Bondy, France.
BMC Genomics. 2017 Sep 1;18(1):681. doi: 10.1186/s12864-017-4076-9.
Thanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging.
In this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy.
We showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit "Epidemiology" in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines.
We have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.
由于经过数百万年进化形成的特殊适应性，高等白蚁共生系统中木质素、纤维素和半纤维素的分解效率超过了许多其他利用木质纤维素的环境。特别是，对其共生微生物的研究应该能揭示有趣的碳水化合物活性酶，这是该行业主要关注的内容。此前的宏转录组学报告（高通量mRNA测序）强调了白蚁后肠消化组中纤维素和半纤维素降解基因的高丰度和过表达，表明该技术在寻找新酶方面的潜力。然而，与材料采样和文库制备步骤相关的几个因素使得对白蚁肠道原核共生体的宏转录组学研究具有挑战性。
在本研究中，我们首先研究了采样策略（包括整个白蚁肠道和肠腔液）对高等白蚁肠道共生细菌多样性和宏转录组图谱的影响。其次，我们评估了两种文库制备流程中结合使用的不同商业试剂盒，以寻找最佳的细菌mRNA富集策略。
我们发现，采样策略对所产生的结果没有显著影响，无论是在微生物的丰度还是其转录组图谱方面。然而，收集肠腔液可减少宿主来源的不需要的RNA种类的共扩增。此外，对于所研究的四种高等白蚁物种，与使用MICROBExpress和MICROBEnrich试剂盒的流程相比，采用Ribo-Zero Gold rRNA去除试剂盒“Epidemiology”与Poly(A) Purist MAG试剂盒相结合的文库制备流程能更有效地去除rRNA和多聚腺苷酸化mRNA（rRNA去除率高达98.44%）。对于所有研究样本，已表明Ribo-Zero和MICROBExpress去除后的基因表达谱与未去除的总RNA测序数据具有高度相关性，这表明所研究的流程没有系统性偏差。
我们广泛评估了采样策略和文库制备步骤对高等白蚁肠道共生细菌宏转录组图谱的影响。所提出的方法学方法在增强高等白蚁肠道菌群的宏转录组学研究以及揭示新型碳水化合物活性酶方面具有巨大潜力。
