Table 1_Gut mycobiome maturation and its determinants during early childhood: a comparison of ITS2 amplicon and shotgun metagenomic sequencing approaches.docx

IntroductionMicrobial colonization of the gut in early life is important for the development of metabolism, immunity, and the brain. Fungi and bacteria both colonize the human infant gut. The relatively smaller contribution of fungi to the gut microbiome, as compared to bacteria, has posed technical challenges for the precise characterization of fungal communities (mycobiomes) and limited the ability to longitudinally examine mycobiome development.
BackgroundThe aims of this study were to (1) characterize mycobiome maturation and identify clinical determinants of mycobiome compositional variation during the first 2 years of life and (2) compare two sequencing approaches (ITS2 amplicon and whole genome metagenomics) for characterizing mycobiome maturational features. Longitudinal fecal samples and associated clinical metadata were obtained from subjects enrolled as part of the MAGIC (Microbiome, Antibiotics and Growth Infant Cohort) study.
ResultsOverall, fungal richness increased and mycobiome composition changed in a similar ordered pattern during the first 2 years of life utilizing either amplicon or metagenomic sequencing approaches. Less resolution of taxa to species and genera levels was observed for the metagenomic dataset. The predominant taxa identified by both sequencing approaches, Candida albicans, Saccharomyces/S. cerevisiae, and Malassezia restricta, each exhibited similar dynamics in abundances and prevalences over the first 2 years of life, irrespective of sequencing approach. Antibiotic exposure and breastfeeding status contributed to time-specific mycobiome compositional variation, results that were consistent for both types of sequence datasets. Candida albicans exhibited altered abundance dynamics in association with perinatal antibiotic exposure and birth mode for both sequencing approaches. Post hoc analyses suggested that the birth mode association could be driven by exposure to perinatal antibiotics in children delivered by cesarean section rather than by birth mode itself.
DiscussionIn summary, amplicon and metagenomic sequencing approaches provide generally similar results with respect to mycobiome maturational dynamics and the contribution of clinical variables to variation. Differences in taxa identification by the two approaches likely due to sequence database differences, primer/genome sequence variation, and/or sequencing depth should be taken into consideration.

引言
生命早期肠道微生物定植对代谢、免疫及大脑发育具有重要意义。真菌与细菌均可定植于人类婴儿肠道。相较于细菌，真菌在肠道微生物组中的相对占比较低，这为真菌群落（mycobiome）的精准表征带来了技术挑战，同时也限制了对其发育进行纵向监测的能力。
背景
本研究的目标为：（1）解析生命最初2年内真菌群落（mycobiome）的成熟过程，并明确真菌群落组成变异的临床影响因素；（2）比较两种测序方法——ITS2扩增子测序与全基因组宏基因组测序——在表征真菌群落成熟特征方面的效果。本研究的受试者来自MAGIC（Microbiome, Antibiotics and Growth Infant Cohort，微生物组、抗生素与婴儿生长队列）研究，获取了其纵向粪便样本及配套的临床元数据。
结果
总体而言，无论采用扩增子测序还是宏基因组测序方法，在生命最初2年内，真菌丰富度均呈上升趋势，真菌群落组成也呈现相似的有序变化模式。宏基因组数据集在分类群鉴定至种、属水平时的分辨能力相对较弱。两种测序方法鉴定出的优势分类群——白色念珠菌、酵母菌/酿酒酵母以及限制性马拉色菌——在生命最初2年内的丰度和流行率动态变化均相似，与测序方法无关。抗生素暴露与母乳喂养状态均会导致特定时间点的真菌群落组成发生变化，这一结果在两类测序数据中均一致。无论采用哪种测序方法，白色念珠菌的丰度动态变化均与围产期抗生素暴露及分娩方式相关。事后分析表明，分娩方式与真菌群落的关联可能是由剖宫产婴儿的围产期抗生素暴露所驱动，而非分娩方式本身。
讨论
综上，针对真菌群落的发育动态以及临床变量对群落变异的影响而言，扩增子测序与宏基因组测序方法总体上可获得相似的结果。两类方法在分类群鉴定上的差异可能源于序列数据库差异、引物/基因组序列变异以及/或测序深度，这些因素均需纳入考量。