利用以基因組為中心的宏基因組學方法從微生物組數據中發現新見解

Utilization of genome-centric metagenomics approach to uncover novel insights from microbiome data

陳煜翔(Yu-Hsiang Chen)

指導教授 ： 盧子彬

共同指導教授 ： 湯森林(Sen-Lin Tang)

國立臺灣大學/電機資訊學院/生醫電子與資訊學研究所/博士(2021年)

https://doi.org/10.6342/NTU202104562

摘要

過去十年，隨著高通量核酸定序和生物資訊學技術的快速發展，宏基因體定序數據已被廣泛用於探索微生物基因體。此高通量核酸定序法解決了傳統微生物學方法難以解決的重大問題，例如，宏基因組霰彈槍定序使我們能推測難以培養的微生物其潛在功能和生理特徵，這些知識無法透過培養其微生物而獲得。
宏基因體技術的發展使我們能從複雜群落中組裝微生物基因體，並研究這些微生物基因體的特徵，這種方法稱為以基因組為中心的宏基因體學。透過宏基因體學，我們能深入了解生態系統中各個微生物的潛在作用。雖然該方法在微生物研究中已提供許多新知識，但與從純培養物中組裝的基因體相比，從微生物群體中組裝的基因體的品質通常較差。本論文應用了多種方法來解決此困境，第1章簡介了當代以基因組為中心的宏基因組學方法的實踐、應用和問題，第2章描述了珊瑚骨骼中Prosthecochloris基因體的組裝，第3章描述了結合短讀長與長讀長定序法以取得星野黑皮海綿中藍綠菌的完整基因體，第4章敘述了應用短讀長與長讀長定序法從複雜微生物群落中取得數個高品質的微生物基因體，第5章總結並討論了此論文的結果。
第2章成功組裝了珊瑚骨骼中Prosthecochloris基因體。先前的研究顯示，有一群綠硫菌－Prosthecochloris－廣泛存在珊瑚骨骼中，其有共通的親緣關係，故被稱為coral-associated Prosthecochloris (CAP)。其可能在維持珊瑚健康方面扮演重要角色。然而，尚未有CAP能被培養，此外從珊瑚骨骼宏基因組中取得的 CAP 基因體的品質很差，使我們難以藉由此基因體分析探索 CAP 的假定功能。因此，在這項研究中，我從珊瑚骨骼培養中重建了高品質的 CAP 基因體，並基於其基因體描述此細菌的特徵進而提出了其可能的共生作用。
第3章為關於星野黑皮海綿中藍綠菌的基因體組裝。短讀長之宏基因組數據的組裝通常使我們獲得高度片段化的序列。高片段化的序列會丟失有關基因順序的訊息並阻礙我們取得高品質的基因體。例如，在以短讀長定序組裝珊瑚殺手星野黑皮海綿的微生物基因體時，我們只能取得碎片化的基因體，並且基因體含有自其他物種的序列污染，此影響了我們對於星野黑皮海綿內藍綠菌的研究。在本論文中，我結合 Nanopore 和 Illumina 定序平台來獲取星野黑皮海綿內藍綠菌的完整基因體。
第4章描述了以Nanopore 和 Illumina 定序平台從複雜微生物群落中取得數個高品質的微生物基因體。星野黑皮海綿內藍綠菌的完整基因體之取得表明了使用混合平台的實用性。然而，從具有復雜微生物群落的宏基因體中取得基因體較為困難。在此，我發展了一種新的分析流程。我結合 Nanopore 和 Illumina 序列，從復雜的微生物群落中取得高品質的微生物基因體。我應用了此新的分析流程，並成功地組裝來自一個不完全對流湖中數百個新的微生物基因體。此結果表明該分析流程的實用性。最後，第5章總結了本論文的結果，並討論各未來研究的方向。

關鍵字

宏基因體

並列摘要

With the rapid development of high-throughput nucleic acid sequencing and bioinformatics techniques in the last decade, metagenomic sequencing data have been widely used to provide novel and significant insights in microbial genomics. The high-throughput sequencing approaches have solved some significant questions, hardly solved by the traditional microbiological approaches. For example, putative functions and physiological features of many difficult-to-culture or fastidious microbes, inaccessible by culture-based methods, have been unveiled by metagenomic shotgun sequencing techniques.
Metagenomic approaches allow us to retrieve metagenome-assembled genomes (MAGs) from complex communities and to study details of genomic features in the MAGs. This method, also called genome-centric metagenomics, provides us with insights into the potential roles of individual microbes in a given ecosystem. Although the approach is unprecedentedly powerful and useful in microbiological studies, the qualities of MAGs recovered from microbiome data are often poor compared to genomes isolated from pure culture. In this dissertation, several methods have been developed and applied to solve the challenges of high-quality genome recovery from raw metagenomic data. Chapter 1 describes the implementation, applications, and challenges of current genome-centric metagenomics. Chapter 2 describes the recovery of high-quality MAGs from enriched coral endolithic culture. Chapter 3 describes the combination of Nanopore and Illumina sequencing platforms to recover a complete genome of dominant cyanobacteria in a coral-killing sponge, Terpios hoshinota. In chapter 4, a workflow that combines Nanopore and Illumina sequencing platforms to recover prokaryotic MAGs from complex microbial communities is depicted.
In Chapter 2, the high-quality genomes of coral-associated Prosthecochloris (CAP) are successfully recovered from coral skeletons. Previous studies revealed that CAP, a genus of green sulfur bacteria, are dominant in coral skeletons and could play a pivotal role in maintaining coral health. However, the CAPs are yet to be cultivated and the quality of a recovered CAP genome from the metagenomes of coral skeletons is poor, hindering the exploration of putative functions of CAP via genomic analyses. Hence, in this study, we reconstruct high-quality CAP genomes from enriched coral endolithic culture and characterize the bacteria and propose putative syntrophic interactions based on genomic analyses.
In Chapter 3, the complete genome of the dominant cyanobacterium in Terpios hoshinota is retrieved. Assembly of short-read metagenomic data usually leads to fragmented sequences. Fragmented sequences lose information on gene order and impede the recovery of high-quality MAGs. For instance, the genome reconstruction of uncultured cyanobacterium dominant in a coral-killing sponge, T. hoshinota, using short-read metagenomic data lead to highly fragmented MAG, and the MAG is vastly contaminated with heterogeneous sequences, compromising the study of the cyanobacterium. In this study, we combine Nanopore and Illumina sequencing platforms to recover a complete genome of the cyanobacterium.
In Chapter 4, a workflow to recover MAGs from complex communities using long reads and short reads is constructed. The success of implementing Nanopore and Illumina sequencing platforms on the recovery of MAG from Terpios hoshinota metagenome, which contains a simple microbial community, indicates the practicality of using hybrid platforms on the genome recovery from metagenomes. However, recovering MAGs from metagenomes with complex microbial communities is more challenging. Here, we developed a novel workflow with combining Nanopore and Illumina sequences to recover high-quality MAGs from complex microbial communities. Hundreds of novel MAGs from a meromictic lake are successfully recovered using this newly-developed workflow, indicating the practicality of the workflow. Lastly, Chapter 5 summarizes and concludes the results of this dissertation and provide future work to further investigate the microbial ecology of these studies.

並列關鍵字

metagenomics ； microbiome ； symbiosis ； metagenome-assembled genome

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