Wunschbaby Institut Feichtinger, Lainzerstrasse 6, Vienna 1130, Austria.
European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK; Department of Anatomy, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
Reprod Biomed Online. 2021 Jun;42(6):1097-1107. doi: 10.1016/j.rbmo.2021.03.016. Epub 2021 Mar 27.
Full-length 16S rRNA gene sequencing using nanopore technology is a fast alternative to conventional short-read 16S rRNA gene sequencing with low initial investment costs that has been used for various microbiome studies but has not yet been investigated as an alternative approach for endometrial microbiome analysis. Is in-situ 16S rRNA gene long-read sequencing using portable nanopore sequencing technology feasible and reliable for endometrial microbiome analysis?
A prospective experimental study based on 33 patients seeking infertility treatment between January and October 2019. A 16S rRNA gene long-read nanopore sequencing protocol for analysing endometrial microbiome samples was established, including negative controls for contamination evaluation and positive controls for bias evaluation. Contamination caused by kit and exterior sources was identified and excluded from the analysis. Endometrial samples from 33 infertile patients were sequenced using the optimized long-read nanopore sequencing protocol and compared with conventional short-read sequencing carried out by external laboratories.
Of the 33 endometrial patient samples, 23 successfully amplified (69.7%) and their microbiome was assessed using nanopore sequencing. Of those 23 samples, 14 (60.9%) were Lactobacillus-dominated (>80% of reads mapping to Lactobacillus), with 10 samples resulting in more than 90% Lactobacillus reads. Our long-read nanopore sequencing revealed results similar to two conventional short-read sequencing approaches and to long-read sequencing validation carried out in external laboratories.
In this pilot study, 16S rRNA gene long-read nanopore sequencing was established to analyse the endometrial microbiome in situ that could be widely applied owing to its cost efficiency and portable character.
使用纳米孔技术进行全长 16S rRNA 基因测序是一种快速替代传统短读长 16S rRNA 基因测序的方法，具有较低的初始投资成本，已被用于各种微生物组研究，但尚未被视为子宫内膜微生物组分析的替代方法。使用便携式纳米孔测序技术进行原位 16S rRNA 基因长读测序，是否可行且可靠，用于子宫内膜微生物组分析？
这是一项基于 2019 年 1 月至 10 月间 33 名寻求不孕治疗的患者的前瞻性实验研究。建立了一种用于分析子宫内膜微生物组样本的 16S rRNA 基因长读纳米孔测序方案，包括用于污染评估的阴性对照和用于偏倚评估的阳性对照。识别并排除了试剂盒和外部来源引起的污染。使用优化的长读纳米孔测序方案对 33 名不孕患者的子宫内膜样本进行测序，并与外部实验室进行的传统短读测序进行比较。
在 33 个子宫内膜患者样本中，有 23 个成功扩增（69.7%），并使用纳米孔测序评估了它们的微生物组。在这 23 个样本中，有 14 个（60.9%）为乳杆菌主导型（>80%的读数映射到乳杆菌），其中 10 个样本的乳杆菌读数超过 90%。我们的长读纳米孔测序结果与两种传统短读测序方法相似，与外部实验室进行的长读测序验证结果也相似。
在这项初步研究中，建立了原位 16S rRNA 基因长读纳米孔测序方法来分析子宫内膜微生物组，由于其成本效益和便携性，该方法可能得到广泛应用。
