Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
复制标题
DOI：
10.1371/journal.pone.0065226
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Hoffman NG
中科院分区：
文献类型：
--
作者：
Salipante SJ;Sengupta DJ;Rosenthal C;Costa G;Spangler J;Sims EH;Jacobs MA;Miller SI;Hoogestraat DR;Cookson BT;McCoy C;Matsen FA;Shendure J;Lee CC;Harkins TT;Hoffman NG
对由复杂的多菌患者标本组成的单个细菌物种进行分类仍然是临床上常用的基于培养和分子微生物学技术的挑战。因此，我们采用了来自元基因组学研究的做法，直接从患者那里快速对临床标本的细菌成分进行分类，而不需要事先培养。我们结合了半导体深度测序协议，产生跨越16S核糖体核糖体基因可变区1和2(∼360bp)的读数，并结合了显著提高无错序列比例的去噪管道。所得到的序列可用于执行准确的属或种级别的分类分配。我们通过深度测序、基于培养的菌株分型和散装PCR产物的Sanger测序来探索具有挑战性的、不同种类的临床标本的微生物组成。我们报道，深度测序可以对混合样本中的细菌物种进行分类，而常规临床方法无法从混合样本中获得有用的数据。对囊性纤维化(CF)患者的痰标本进行深度测序，发现在标准临床培养方法无法检测到的标本中，尤其是低流行率或挑剔的细菌，囊性纤维化病原体的描述很好。我们还发现，根据呼吸道微生物区系的系统发育组成，提交进行CF诊断检查的痰可分为有限数量的组，这表明元基因组图谱可能在未来被证明是一种有用的临床诊断策略。所描述的方法足够快速(理论上与当天的周转时间兼容)，并且对于常规临床使用来说是廉价的。
Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.
登录
查看更多内容
影响因子：
3.7
作者：
Jünemann S;Prior K;Szczepanowski R;Harks I;Ehmke B;Goesmann A;Stoye J;Harmsen D
通讯作者：
Harmsen D
影响因子：
11
作者：
Guss, Adam M.;Roeselers, Guus;Cavanaugh, Colleen M.
通讯作者：
Cavanaugh, Colleen M.
影响因子：
4.4
作者：
Ashelford, KE;Chuzhanova, NA;Weightman, AJ
通讯作者：
Weightman, AJ
影响因子：
56.9
作者：
Gill, Steven R.;Pop, Mihai;Nelson, Karen E.
通讯作者：
Nelson, Karen E.
影响因子：
14.9
作者：
Cole JR;Wang Q;Cardenas E;Fish J;Chai B;Farris RJ;Kulam-Syed-Mohideen AS;McGarrell DM;Marsh T;Garrity GM;Tiedje JM
通讯作者：
Tiedje JM