Virtanen Seppo, Saqib Schahzad, Kanerva Tinja, Ventin-Holmberg Rebecka, Nieminen Pekka, Holster Tiina, Kalliala Ilkka, Salonen Anne
Department of Obstetrics and Gynaecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
Faculty of Medicine, Human Microbiome Research Program, University of Helsinki, Helsinki, Finland.
Microbiome. 2024 Dec 28;12(1):273. doi: 10.1186/s40168-024-01993-9.
Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial communities. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction-based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing.
Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment-guided tool provided ecosystem-specific bacterial annotations that were confirmed by shotgun metagenomic sequencing (VIRGO, MetaPhlAn, Kraken2). Fungi were identified in 39/50 samples with ITS sequencing while in the metagenome data fungi largely remained undetected due to their low abundance and database issues. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.
Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Conventional metagenomic sequencing does not provide sufficient fungal genome coverage for their reliable detection in vaginal samples. Text extraction-based annotation tool facilitates ecosystem-specific characterization and interpretation of microbial communities by coupling sequence homology to microbe metadata readily available through public databases. Video Abstract.
对特定类群标签进行扩增子测序，如用于细菌的16S rRNA基因和用于真菌的内转录间隔区（ITS）区域，被广泛用于研究微生物群落。到目前为止，大多数人体研究都集中在细菌上，而关于健康和疾病中与宿主相关真菌的研究直到最近才开始积累。为了能够对人体和环境样本中的细菌和真菌群落进行经济高效的平行分析，我们开发了一种方法，将16S rRNA基因和ITS1扩增子混合在一起，用于单次Illumina MiSeq或HiSeq运行，并在基于引物的分离后进行分析。使用Blast结合基于文本提取的迭代过滤方法进行分类学注释，该方法利用公共数据库中的大量文献记录来选择最可能的匹配项，并通过鸟枪法宏基因组测序进一步验证。
使用50份阴道样本，我们表明联合运行在细菌组成和多样性方面提供了与传统16S rRNA基因扩增子测序相当的结果。基于文本提取的分类学注释引导工具提供了特定生态系统的细菌注释，这些注释通过鸟枪法宏基因组测序（VIRGO、MetaPhlAn、Kraken2）得到了证实。通过ITS测序在39/50份样本中鉴定出了真菌，而在宏基因组数据中，由于真菌丰度低和数据库问题，真菌大多未被检测到。健康女性阴道生态系统中细菌和真菌的共丰度分析未显示出强烈的跨类群相关性。
细菌和真菌的联合扩增子测序为同时分析同一样本中的微生物群和真菌群提供了一种简单且经济高效的方法。传统的宏基因组测序在阴道样本中无法提供足够的真菌基因组覆盖范围以进行可靠检测。基于文本提取的注释工具通过将序列同源性与可通过公共数据库轻松获取的微生物元数据相结合，促进了对微生物群落的特定生态系统特征描述和解释。视频摘要。
