MinION 测序结直肠癌肿瘤微生物组-与扩增子测序和 RNA 测序的比较。
MinION Sequencing of colorectal cancer tumour microbiomes-A comparison with amplicon-based and RNA-Sequencing.
机构信息
Department of Surgery, University of Otago, Christchurch, New Zealand.
Biostatistics and Computational Biology Unit, University of Otago, Christchurch, New Zealand.
出版信息
PLoS One. 2020 May 20;15(5):e0233170. doi: 10.1371/journal.pone.0233170. eCollection 2020.
BACKGROUND
Recent evidence suggests a role for the gut microbiome in the development and progression of many diseases and many studies have been carried out to analyse the microbiome using a variety of methods. In this study, we compare MinION sequencing with meta-transcriptomics and amplicon-based sequencing for microbiome analysis of colorectal tumour tissue samples.
METHODS
DNA and RNA were extracted from 11 colorectal tumour samples. 16S rRNA amplicon sequencing and MinION sequencing was carried out using genomic DNA, and RNA-Sequencing for meta-transcriptomic analysis. Non-human MinION and RNA-Sequencing reads, and 16S rRNA amplicon sequencing reads were taxonomically classified using a database built from available RefSeq bacterial and archaeal genomes and a k-mer based algorithm in Kraken2. Concordance between the three platforms at different taxonomic levels was tested on a per-sample basis using Spearman's rank correlation.
RESULTS
The average number of reads per sample using RNA-Sequencing was greater than 129 times that generated using MinION sequencing. However, the average read length of MinION sequences was more than 13 times that of RNA or 16S rRNA amplicon sequencing. Taxonomic assignment using 16S sequencing was less reliable beyond the genus level, and both RNA-Sequencing and MinION sequencing could detect greater numbers of phyla and genera in the same samples, compared to 16S sequencing. Bacterial species associated with colorectal cancer, Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis and Porphyromonas gingivalis, were detectable using MinION, RNA-Sequencing and 16S rRNA amplicon sequencing data.
CONCLUSIONS
Long-read sequences generated using MinION sequencing can compensate for low numbers of reads for bacterial classification. MinION sequencing can discriminate between bacterial strains and plasmids and shows potential as a cost-effective tool for rapid microbiome sequencing in a clinical setting.
背景
最近的证据表明，肠道微生物群在许多疾病的发生和发展中起作用，许多研究已经使用各种方法来分析微生物群。在这项研究中，我们比较了 MinION 测序与宏转录组学和基于扩增子的测序，以分析结直肠肿瘤组织样本的微生物组。
方法
从 11 个结直肠肿瘤样本中提取 DNA 和 RNA。使用基因组 DNA 进行 16S rRNA 扩增子测序和 MinION 测序，并进行 RNA-Seq 进行宏转录组分析。使用基于可用 RefSeq 细菌和古菌基因组的数据库和 Kraken2 中的 k-mer 算法对非人类 MinION 和 RNA-Seq 读数以及 16S rRNA 扩增子测序读数进行分类。在逐个样本的基础上，使用 Spearman 秩相关检验测试三个平台在不同分类水平上的一致性。
结果
使用 RNA-Seq 获得的每个样本的平均读数数是使用 MinION 测序获得的平均读数数的 129 倍以上。然而，MinION 序列的平均读长是 RNA 或 16S rRNA 扩增子测序的 13 倍以上。16S 测序在属级以上的分类可靠性较低，与 16S 测序相比，RNA-Seq 和 MinION 测序可以在相同的样本中检测到更多的门和属。与结直肠癌相关的细菌物种，包括具核梭杆菌、微小 Parvimonas、脆弱拟杆菌和牙龈卟啉单胞菌，可使用 MinION、RNA-Seq 和 16S rRNA 扩增子测序数据检测到。
结论
使用 MinION 测序生成的长读序列可以弥补细菌分类的低读数数量。MinION 测序可以区分细菌菌株和质粒，并显示出作为一种在临床环境中快速微生物组测序的具有成本效益的工具的潜力。
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