Anaerobic digestion (AD) has long been critical technology for green energy, but the majority of the microorganisms involved are unknown and are currently not cultivable, which makes abundance tracking difficult. Developments in nanopore long-read sequencing make it a promising approach for monitoring microbial communities via metagenomic sequencing. For reliable monitoring of AD via long reads, we established a robust protocol for obtaining less fragmented, high-quality DNA, while preserving bacteria and archaea composition, for a broad range of different biogas reactors. Samples from 20 different biogas/wastewater reactors were investigated, and a median of 20.5 Gb sequencing data per nanopore flow cell was retrieved for each reactor using the developed DNA isolation protocol. The nanopore sequencing data were compared against Illumina sequencing data while using different taxonomic indices for read classifications. The Genome Taxonomy Database (GTDB) index allowed sufficient characterisation of the abundance of bacteria and archaea in biogas reactors with a dramatic improvement (1.8- to 13-fold increase) in taxonomic classification compared to the RefSeq index. Both technologies performed similarly in taxonomic read classification with a slight advantage for Illumina in regard to the total proportion of classified reads. However, nanopore sequencing data revealed a higher genus richness after classification. Metagenomic read classification via nanopore provides a promising approach to monitor the abundance of taxa present in a microbial AD community as an alternative to 16S ribosomal RNA studies or Illumina Sequencing.

中文翻译：

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通过对20种不同沼气/废水植物样品中复杂微生物群落的长期阅读纳米孔测序进行的丰度跟踪

厌氧消化（AD）长期以来一直是绿色能源的关键技术，但是涉及的大多数微生物是未知的，目前尚无法培养，这使得很难进行丰度追踪。纳米孔长测序技术的发展使其成为一种通过宏基因组测序监测微生物群落的有前途的方法。为了通过长时间读取对AD进行可靠的监控，我们建立了一个健壮的协议，可为各种不同的沼气反应器获取较少碎片的高质量DNA，同时保留细菌和古细菌的成分。研究了来自20个不同沼气/废水反应器的样品，并使用开发的DNA分离方案为每个反应器检索了每个纳米孔流动池20.5 Gb测序数据的中位数。将纳米孔测序数据与Illumina测序数据进行比较，同时使用不同的分类索引进行读取分类。基因组分类数据库（GTDB）索引可以充分表征沼气反应器中细菌和古细菌的数量，与RefSeq索引相比，分类学分类具有显着改善（增加了1.8到13倍）。两种技术在分类读物分类中的表现相似，对Illumina而言，分类读物的总比例略有​​优势。然而，纳米孔测序数据显示分类后具有较高的属丰富度。通过纳米孔进行的元基因组阅读分类提供了一种有前途的方法，可监测微生物AD群落中存在的分类单元的丰富度，以替代16S核糖体RNA研究或Illumina测序。