Chen Arnold, Nchinda Nkazi, Cira Nate J
Meinig School of Biomedical Engineering, Cornell University, Ithaca, 14853, NY, USA.
Harvard Medical School, Harvard University, Boston, 02115, MA, USA.
Microb Genom. 2025 Mar;11(3). doi: 10.1099/mgen.0.001378.
The sequence of the 16S region is taxonomically informative and widely used for genotyping microbes. While it is easy and inexpensive to genotype several isolates by Sanger sequencing the 16S region, this method becomes quite costly if scaled to many isolates. High-throughput sequencing provides one potential avenue for obtaining 16S sequences at scale but presents additional challenges. First, DNA purification workflows for high-throughput sample preparation are labour-intensive and expensive. Second, cost-effective multiplexing and library preparation schemes are difficult to implement for many libraries on a single sequencing run. Therefore, we implemented a scalable protocol for isolate genotyping involving colony polymerase chain reaction (PCR) with simple cell lysis as well as a four-barcode indexing scheme that enables scalable multiplexing and streamlined library preparation by amplifying with four primers simultaneously in a single reaction. We tested this protocol on 93 colonies cultured from environmental samples, and we were able to ascertain the identity of ~90% of microbial isolates.
16S区域的序列具有分类学信息，被广泛用于微生物基因分型。虽然通过桑格测序法对多个分离株的16S区域进行基因分型既简单又便宜，但如果扩大到许多分离株，这种方法成本就会相当高。高通量测序为大规模获取16S序列提供了一条潜在途径，但也带来了额外挑战。首先，用于高通量样品制备的DNA纯化工作流程既费力又昂贵。其次，对于许多文库在一次测序运行中实施具有成本效益的多重化和文库制备方案很困难。因此，我们实施了一种用于分离株基因分型的可扩展方案，该方案涉及采用简单细胞裂解的菌落聚合酶链反应（PCR）以及一种四条形码索引方案，该方案通过在单个反应中同时用四种引物进行扩增，实现可扩展的多重化和简化的文库制备。我们在从环境样品中培养的93个菌落上测试了该方案，并且能够确定约90%的微生物分离株的身份。
