技术说明:使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。
Technical note: overcoming host contamination in bovine vaginal metagenomic samples with nanopore adaptive sequencing.
机构信息
Centre for Animal Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Queensland 4072, Australia.
Faculty of Science, School of Veterinary Science, The University of Queensland, Queensland 4072, Australia.
出版信息
J Anim Sci. 2022 Jan 1;100(1). doi: 10.1093/jas/skab344.
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.
动物宏基因组学研究,其中对宿主相关微生物组进行了分析,对我们理解家畜的生理功能、健康和疾病易感性做出了越来越重要的贡献。这些研究中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效果的研究。我们比较了牛津纳米孔技术(ONT)自适应测序的新方法,该方法可用于靶向或消除特定的遗传序列,与标准 ONT 测序、Illumina 16S rDNA 扩增子测序和 Illumina 鸟枪法测序进行比较。评估了每种方法在回收宏基因组数据和回忆宏基因组图谱方面的效率。与其他方法相比,ONT 自适应测序每 1 Gb 序列数据产生的宏基因组数据量更高。ONT 自适应测序的测序效率提高,相应减少了高宿主与微生物 DNA 比例的宏基因组样本提供足够覆盖所需的原始数据量。此外,ONT 自适应测序产生的长读长保留了读信息的连续性,这有利于对宏基因组的分类学和功能图谱进行深入注释。不同的方法导致鉴定出不同的分类单元。使用 16S rDNA 扩增子测序方法时,丰度较低且归类于未分类梭菌目(Order "Unclassified Clostridiales")的属梭菌(Clostridium),在使用其他三种方法测序时被鉴定为样本中的优势属。此外,ONT 自适应测序鉴定出的注释基因数量更多,并且对大多数常见注释基因的覆盖度也很高。本研究说明了 ONT 自适应测序在提高高宿主与微生物 DNA 比例的微生物组样本中获得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。