Bigdeli Saharnaz, Dettloff Roger O, Frank Curtis W, Davis Ronald W, Crosby Laurel D
Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America; Department of Chemical Engineering, Stanford University, Stanford, CA, United States of America.
Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, United States of America; Caerus Molecular Diagnostics, Mountain View, CA, United States of America.
PLoS One. 2015 Feb 17;10(2):e0117738. doi: 10.1371/journal.pone.0117738. eCollection 2015.
Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.
微滴是分离单个细胞和扩增DNA的有效平台。然而，一个关键挑战是回收单个微滴的内容物用于下游分析。本文提供了一种将细胞嵌入海藻酸盐微球并对分离出的细胞进行多次连续操作的方法。将球形红细菌细胞稀释在海藻酸盐聚合物中，并用指尖气溶胶喷雾器喷入微滴中。将包封的细胞裂解，然后进行常规PCR，或使用多重置换扩增（MDA）或两步PCR方案进行全基因组扩增。PCR后的显微镜检查表明，被占据微球的内腔含有荧光染色的DNA产物，但用phi29进行多重置换扩增仅产生少量聚合酶菌落。两步全基因组扩增方案成功产生了荧光物质，从微球等分试样中提取的DNA进行定量PCR表明，微球内的拷贝数扩增了多达3个数量级。通过稀释系列对含有荧光物质的微球进行分选，并用荧光酶标仪进行筛选以鉴定单个微球。从单个分离物中提取DNA，用全长测序接头重新扩增，然后使用Illumina MiSeq平台对单个分离物进行测序。过滤读数后，唯一在NCBI核苷酸数据库中与基因组集体匹配的序列属于球形红细菌。这表明无需培养即可从单个微球的内容物中生成可用于测序的DNA。然而，两步全基因组扩增策略在低基因组覆盖率和全基因组读数频率分布不均方面存在局限性。本文提供了一种将细胞嵌入海藻酸盐微球并在常见的批量反应中对分离出的细胞进行PCR的简单方法，尽管必须进一步开展工作以提高单基因组的扩增覆盖率。
