RNA-Seq Analysis to confirm Different Transcriptomic Profiles of Soil-Derived Functional Module Consortia.

Purpose: Deconstructing the soil microbiome into reduced-complexity functional modules represents a novel method of microbiome analysis. The goals of this study are to confirm differences in transcriptomic patterns among five functional module consortia. Methods: mRNA profiles of 3 replicates each of functional module enrichments of soil inoculum in M9 media with either 1) xylose, 2) n-acetylglucosamine, 3) glucose and gentamycin, 4) xylan, or 5) pectin were generated by sequencing using an Illumina platform (GENEWIZ performed sequencing). Sequence reads that passed quality filters were aligned to a soil metagenome using Burrows Wheeler Aligner. Resulting SAM files were converted to raw reads using HTSeq, and annotated using Uniref90 or EGGNOG databases. Results: To reduce the size of the RNA-Seq counts table and increase its computational tractability, transcripts containing a minimum of 75 total counts, but no more than 3 zero counts, across the 15 samples were removed. The subsequent dataset was normalized using DESeq2, resulting in a dataset consisting of 6947 unique transcripts across the 15 samples, and 185,920,068 reads. We identified gene categories that were enriched in a sample type relative to the overall dataset using Fisher's exact test. Conclusions: our dataset confirms that the functional module consortia generated from targeted enrichments of a starting soil inoculum had distinct functional trends by enrichment type. Overall design: mRNA profiles of enriched functional consortia were sequenced using an Illumina HiSeq platform.

研究目的：将土壤微生物组拆解为低复杂度功能模块，是一种全新的微生物组分析方法。本研究旨在验证5种功能模块菌群的转录组模式差异。
研究方法：以M9培养基为基底，对土壤接种物进行功能模块富集，共设置5种底物条件，分别为1）木糖、2）N-乙酰葡糖胺、3）葡萄糖与庆大霉素、4）木聚糖、5）果胶，每组设置3次生物学重复。采用Illumina平台（测序服务由GENEWIZ提供）对各组样本的信使RNA（mRNA）进行测序。将通过质量过滤的序列读段通过Burrows-Wheeler Aligner（伯罗斯-惠勒比对器）比对至土壤宏基因组，随后利用HTSeq将生成的SAM文件转换为原始读段，并通过Uniref90或EGGNOG数据库完成功能注释。
研究结果：为缩减RNA测序（RNA-Seq）计数表的规模并提升其计算可处理性，本研究移除了在全部15个样本中总计数不少于75且零计数不超过3个的转录本。随后使用DESeq2对剩余数据集进行标准化处理，最终得到涵盖15个样本、包含6947个独特转录本以及185,920,068条序列读段的数据集。本研究采用费希尔精确检验（Fisher's exact test），筛选出相较于整体数据集在特定样本类型中显著富集的基因类别。
研究结论：本数据集证实，通过对初始土壤接种物进行靶向富集得到的功能模块菌群，其功能特征会随富集底物类型的不同而呈现显著差异。
实验整体设计：采用Illumina HiSeq平台对富集获得的功能菌群进行mRNA转录组测序。