We recently developed a high-throughput functional genomics approach, named ‘SorTn-seq’, to identify factors affecting expression of any gene of interest in bacteria. Our approach facilitates high-throughput screening of complex mutant pools, a task previously hindered by a lack of suitable techniques. SorTn-seq combines high-density, Tn5-like transposon mutagenesis with fluorescence-activated cell sorting of a strain harboring a promoter-fluorescent reporter fusion, to isolate mutants with altered gene expression. The transposon mutant pool is sorted into different bins on the basis of fluorescence, and mutants are deep-sequenced to identify transposon insertions. DNA is prepared for sequencing by using commercial kits augmented with custom primers, enhancing ease of use and reproducibility. Putative regulators are identified by comparing the number of insertions per genomic feature in the different sort bins, by using existing bioinformatic pipelines and software packages. SorTn-seq can be completed in 1–2 weeks and requires general microbiology skills and basic flow cytometry experience.

我们最近开发了一种名为“SorTn-seq”的高通量功能基因组学方法，以识别影响细菌中任何感兴趣基因表达的因素。我们的方法有助于对复杂的突变体库进行高通量筛选，这项任务以前因缺乏合适的技术而受到阻碍。SorTn-seq 结合了高密度、Tn 5类转座子诱变，对具有启动子-荧光报告基因融合的菌株进行荧光激活细胞分选，以分离基因表达改变的突变体。转座子突变池根据荧光被分类到不同的 bin 中，并对突变体进行深度测序以识别转座子插入。使用带有定制引物的商业试剂盒准备 DNA 进行测序，从而提高易用性和可重复性。通过使用现有的生物信息学管道和软件包，通过比较不同分类箱中每个基因组特征的插入数量来识别推定的调节器。SorTn-seq 可在 1-2 周内完成，需要具备一般微生物学技能和基本的流式细胞术经验。