Introduction

Metagenomics is increasingly considered for clinical diagnostics. In order for this technology to become integrated in the clinical microbiology laboratory, process controls are required. Molecular diagnostic tests typically integrate an internal control (IC) to detect potential sources of variation and technical failure. However, few studies report on the integration of an IC in metagenomics.

Aim

We aimed to develop an easy-to-use IC method for the process control of library preparation and sequencing applied to metagenomics in clinical microbiology diagnostics using Thermus thermophilus DNA.

Methodology

DNA was extracted from urine samples and sequenced on the Ion Torrent Proton in the absence and presence of incremental concentrations (0.5–2-5%) of IC. Between aliquots of each sample, we compared the IC relative abundance (RA), and after in silico subtraction of IC reads, analysed microbial composition and the RA of pathogens. The optimal IC concentration was defined as the lowest concentration still detectable in all samples with the smallest impact on the microbial composition.

Results

The RA of IC correlated linearly with the spiked IC concentration (r² = 0.99). IC added in a concentration of 0.5% of the total DNA concentration was detectable in all sample aliquots, regardless of human-bacterial DNA proportion, and after in silico removal gave the smallest difference in RA of pathogens compared to the sample aliquot sequenced in the absence of IC. The microbial composition in the presence and absence of IC was highly similar after in silico removal of IC reads (median BC-dissimilarity per sample: 0.059), provided samples had a mean of >10,000 bacterial reads.

Conclusion

T. thermophilus DNA at a percentage of 0.5% of the total DNA concentration was successfully applied for the process control of metagenomics of urine samples. We demonstrated negligible alterations in sample microbial composition after in silico subtraction of IC reads. This approach contributes toward implementation of metagenomics in the clinical microbiology laboratory.

中文翻译：

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嗜热栖热菌DNA可用作内部对照，用于监测尿液样本的临床宏基因组下一代测序的过程。

介绍

元基因组学被越来越多地用于临床诊断。为了使该技术集成到临床微生物学实验室中，需要过程控制。分子诊断测试通常会集成一个内部控制（IC），以检测潜在的变异和技术故障源。但是，很少有研究报道IC在宏基因组学中的集成。

目标

我们的目标是开发一种易于使用的IC方法，用于程序库制备和测序的过程控制，该方法应用于使用嗜热菌（Thermus thermophilus） DNA进行临床微生物学诊断的宏基因组学。

方法

从尿液样品中提取DNA并在不存在和存在递增浓度（0.5–2-5％）的IC的情况下，在离子洪流质子上进行测序。在每个样品的等分试样之间，我们比较了IC相对丰度（RA），并在计算机上扣除了IC读数后，对微生物组成和病原体RA进行了分析。最佳IC浓度定义为在所有样品中仍可检测到的最低浓度，对微生物成分的影响最小。

结果

IC的RA与加标的IC浓度线性相关（r ²  = 0.99）。不论人类细菌DNA的比例如何，在所有样品等分试样中都可以检测到以总DNA浓度的0.5％浓度添加的IC，并且与不进行测序的样品等分试样相比，在计算机去除后，病原体的RA差异最小。 IC。在计算机上通过计算机去除IC读数后，存在和不存在IC的微生物组成高度相似（每个样品的平均BC差异中位数：0.059），前提是样品的平均细菌读数大于10,000。

结论

占总DNA浓度0.5％的嗜热链球菌DNA成功应用于尿液样品宏基因组学的过程控制。我们证明了在计算机对IC读数进行减法运算后，样品微生物组成的变化可忽略不计。这种方法有助于在临床微生物学实验室中实施宏基因组学。