基于rDNA靶向测序和鸟枪法测序相结合的致病菌筛查

Pathogen screening based on a combination of rDNA targeted sequencing and shotgun sequencing

目的基于高通量测序技术,建立靶向测序和鸟枪法测序相结合的致病菌筛查方法,以期用于临床感染标本中可疑致病菌的快速检测。为临床治疗提供参考。方法通过计算机模拟建立分析策略并研究其可行性,然后通过已知细菌进行验证;应用该方法对临床样品进行检测,分析样品中可疑的致病菌。结果在计算机模拟分析的四组标本中,相对应的细菌种属的reads检出比例在总细菌reads数中均为最高。在用已知细菌进行验证的结果中,四种细菌相对应的属reads比例均达到标本总reads数的88%以上,种reads比例均达到标本总reads数的51%以上;三个真菌标本中相对应的真菌属reads比例均达到标本总reads数的91%以上,种reads比例均达到标本总reads数的40%以上。四个细菌样本和三个真菌样本相对应的细菌和真菌属reads和种reads检出比例分别是对应样本中含量最高的属和种,与实际操作的细菌投入量基本相符。计算机模拟分析和用已知细菌测试结果表明,利用16S/ITS全长序列扩增产物进行打断建库可用于细菌和真菌的检测。将rDNA靶向测序和鸟枪法测序相结合,在临床脑脊液中检出红球菌和假单胞菌,在血液和唾液中检出金黄色葡萄球菌。结论靶向测序提高了检测的灵敏性,rDNA靶向测序和鸟枪法测序结果相互印证,提高了病原体检测的准确性。两种测序方法相结合用于病原体检测更加合理有效。

Objective To devise a method of screening for pathogens in clinical specimens through a combination of target sequencing and shotgun sequencing.The method was used to detect potential pathogens in clinical samples in order to provide information for treatment of infections. Methods An analysis algorithm was devised and used on simulated data,and then the analysis algorithm and methodology were validated using sequencing data from known bacteria.This method was then applied to sequencing data from clinical samples. Results The highest percentage of species-specific reads as a proportion of all bacterial reads was obtained in four samples generated by a computer simulation.During validation with known bacteria,the percentage of reads for 4 genera of bacteria in all samples was greater than 88%.The percentage of reads for specific species was greater than 51%.In three fungal samples,the percentage of reads for specific genera was greater than 91% and the percentage of reads for specific species was greater than 40%.Genera and species found in the four bacterial samples and three fungal samples accounted for the highest percentage of reads for genera and species of bacteria and fungi,so the results of analysis were basically consistent with the actual genera and species present.Both simulation data and experimental data with known bacteria indicated that the analysis algorithm and methodology can effectively detect target pathogens using short reads generated as a result of amplification of the full-length sequence of 16 S/ITS.The combination of rDNA targeted sequencing and shotgun sequencing successfully detected the genera Rhodococcus and Pseudomonas in cerebrospinal fluid（CSF）samples and Staphylococcus aureus in blood and saliva samples. Conclusion Targeted sequencing improves the sensitivity of pathogen detection.rDNA targeted sequencing and shotgun sequencing are mutually validating.Combining rDNA targeted sequencing and shotgun sequencing improves the accuracy of pathogen detection.The current results demonstrated that a combination of rDNA targeted sequencing and shotgun sequencing is an effective way to screen for pathogens in clinical samples.