Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium, which was identified at low abundances and categorized under Order “Unclassified Clostridiales” when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations.

中文翻译：

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技术说明：利用纳米孔自适应测序克服牛阴道宏基因组样本中的宿主污染


动物宏基因组研究对宿主相关微生物组进行了分析，对我们了解牲畜的生理功能、健康和疾病易感性做出了越来越重要的贡献。这些研究的主要挑战之一是宿主 DNA 污染，它限制了宏基因组内容的测序能力并降低了宏基因组分析的准确性。这是第一项比较不同测序方法对牛阴道宏基因组样本进行分析的有效性的研究。我们将 Oxford Nanopore Technologies (ONT) 自适应测序的新方法与标准 ONT 测序、Illumina 16S rDNA 扩增子测序和 Illumina 鸟枪法测序进行了比较，该方法可用于靶向或消除已定义的基因序列。评估了每种方法恢复宏基因组数据和调用宏基因组图谱的效率。每 1 Gb 序列数据，ONT 自适应测序比其他方法产生更多的宏基因组数据。 ONT 自适应测序提高了测序效率，从而减少了为具有高宿主与微生物 DNA 比率的宏基因组样本提供足够覆盖所需的原始数据量。此外，ONT自适应测序生成的长读段保留了读段信息的连续性，这有利于对宏基因组的分类学和功能谱进行深入注释。不同的方法导致了不同类群的识别。使用 16S rDNA 扩增子测序方法时，梭菌属被鉴定为低丰度，并被归类为“未分类梭菌目”，而当使用其他三种方法进行测序时，被鉴定为样品中的优势属。 此外，通过 ONT 自适应测序鉴定出更多数量的注释基因，这也对大多数常见注释基因产生了高覆盖率。这项研究说明了 ONT 自适应测序在提高宿主与微生物 DNA 比例高的微生物组样本中的宏基因组数据量方面的优势，以及长读长在保留完整信息以进行准确注释方面的优势。