Ultra-accurate microbial amplicon sequencing with synthetic long reads.
Ultra-accurate microbial amplicon sequencing with synthetic long reads.
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具有合成长读数的超精确微生物扩增子测序。
DOI：
10.1186/s40168-021-01072-3
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发表时间：
2021-06-05
期刊：
影响因子：
15.5
通讯作者：
Yehezkel TB
中科院分区：
文献类型：
--
作者：
Callahan BJ;Grinevich D;Thakur S;Balamotis MA;Yehezkel TB
在已知的许多致病细菌物种中，只有一小部分可以使用标准的下一代DNA测序从复杂的微生物群落中直接识别出来。长读测序提供了鉴定更广泛的物种和在一个物种内区分菌株的潜力，但是在复杂的宏基因组中获得足够的准确性仍然是一个挑战。在这里，我们描述并分析验证了LoopSeq，这是一种市售的合成长读（SLR）测序技术，可以从标准短读产生高度精确的长读。LoopSeq读数足够长且准确，可以直接从复杂样品中识别微生物基因和物种。LoopSeq完全恢复了合成微生物群落中已知菌株的16S rRNA基因的全部多样性。全长LoopSeq reads的每碱基错误率为0.005%，超过了其他长读测序技术报道的准确率。真菌和细菌分离物的18S-ITS和基因组测序证实，LoopSeq测序在长度达6 kb的reads中保持准确性。LoopSeq全长16S rRNA读数可以准确地将零售肉类样品的冲洗液中的生物分类到物种水平，并且可以区分疾病预防控制中心确定为潜在食源性病原体的物种内的菌株。与使用LoopSeq实现的标准Illumina扩增子测序相比，长度和精度的数量级提高使得能够从复杂到低生物量的微生物组样品中进行准确的物种水平和菌株鉴定。使用标准短读测序仪生成准确和长的微生物组测序读数的能力将加速高质量微生物序列数据库的建立，并消除精确微生物基因组学道路上的一个重大障碍。视频摘要。在线版本包含补充材料，可在10.1186/s40168-021- 01073 -3获得。
Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics. Video abstract. The online version contains supplementary material available at 10.1186/s40168-021-01072-3.
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影响因子：
48
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Callahan BJ;McMurdie PJ;Rosen MJ;Han AW;Johnson AJ;Holmes SP
通讯作者：
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