Pu Dan, Qi Yuhua, Cui Lunbiao, Xiao Pengfeng, Lu Zuhong
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, Jiangsu 210009, China.
Anal Chim Acta. 2014 Dec 10;852:274-83. doi: 10.1016/j.aca.2014.09.009. Epub 2014 Sep 16.
We propose a real-time decoding sequencing strategy in which a template is determined without directly measuring base sequence but by decoding two sets of encodings obtained from two parallel sequencing runs. This strategy relies on adding a mixture of different two-base pair, A+G, C+T, A+C, G+T, A+T or C+G (abbreviated as AG, CT, AC, GT, AT, or CG), into the reaction each time. When a template is cyclically interrogated twice with any two kinds of dual mononucleotide addition (AG/CT, AC/GT, and AT/CG), two sets of encodings are obtained sequentially. The two sets of encodings allow for the bases to be sequentially decoded, moving from first to last, in a deterministic manner. This strategy applies fewer cycles to obtain longer read length compared to the traditional real-time sequencing strategy. Partial rnpB gene was applied to verify the applicability of the decoding strategy via pyrosequencing. The results indicated that the sequence could be reconstructed by decoding two sets of encodings. Moreover, streptococcal strains could be differentiated by comparing signal intensity in each cycle and encoding size of each template. This strategy is likely to be applied to differentiate nucleic acid sequence as encoding size and signal intensity in each cycle vary with the base size and composition. Furthermore, it has the potential in building a promising strategy that could be utilized as an alternative to conventional sequencing systems.
我们提出了一种实时解码测序策略，该策略不是直接测量碱基序列，而是通过解码从两次平行测序运行中获得的两组编码来确定模板。此策略依赖于每次向反应中添加不同的两个碱基对的混合物，即A+G、C+T、A+C、G+T、A+T或C+G（简称为AG、CT、AC、GT、AT或CG）。当用任意两种双单核苷酸添加物（AG/CT、AC/GT和AT/CG）对模板进行两轮循环询问时，会依次获得两组编码。这两组编码使得碱基能够以确定的方式从第一个到最后一个依次解码。与传统的实时测序策略相比，该策略应用较少的循环就能获得更长的读长。通过焦磷酸测序应用部分rnpB基因来验证解码策略的适用性。结果表明，可以通过解码两组编码来重建序列。此外，通过比较每个循环中的信号强度和每个模板的编码大小，可以区分链球菌菌株。由于每个循环中的编码大小和信号强度会随碱基大小和组成而变化，因此该策略可能适用于区分核酸序列。此外，它有潜力构建一种有前景的策略，可作为传统测序系统的替代方案。
