Algorithms for Optimal Base-Calling in Sequencing-by-Synthesis

描述（由申请人提供）：下一代合成测序平台实现快速和负担得起的DNA测序。然而，它们获得的读取长度仍然比昂贵的Sanger测序提供的读取长度短，而且它们的准确性不足以用于大多数医学研究。为了确定DNA片段中核苷酸的顺序，合成测序依赖于片段上互补链的酶合成。该合成是通过自由核苷酸的顺序添加实现的；互补链的延伸与DNA片段的第一个未配对碱基的沃森-克里克补体的光学检测。然而，测序单个DNA分子所产生的信号是微弱的，因此它的检测需要复杂和昂贵的硬件。基于集成的系统提供了一种有效的替代方案：它们通过并行地对大量相同的DNA片段拷贝进行测序来放大信号。为了充分获得多个信号源的好处，互补链的延伸应该以相同的速率进行（这样信号就会同步增加）。然而，由于一些链的核苷酸结合偶尔失败，以及其他链的过早延伸，在一个整体中链的合成会不同步。这些所谓的相位效应，本质上是概率性的，限制了合成测序的可实现精度和读取长度。该项目的目标是开发实用的算法，以优化合成测序系统中的碱基调用，提高其有效的读取长度和准确性。为此，我们依靠信号处理和信息论中的概念和工具。我们解决两种广泛使用的系统：Illumina的四色平台和罗氏（454生命科学）焦磷酸测序平台。根据初步结果，如果成功，我们的研究将对需要高性能DNA测序的各种应用产生直接影响。

DESCRIPTION (provided by applicant): Next generation sequencing-by-synthesis platforms enable fast and affordable DNA sequencing. However, read-lengths that they achieve are still shorter than those provided by the costly Sanger sequencing, and their accuracy is insufficient for most medical studies. To determine the order of nucleotides in a DNA fragment, sequencing-by-synthesis relies on enzymatic synthesis of the complementary strand on the fragment. The synthesis is enabled by a sequential addition of free nucleotides; extension of the complementary strand with the Watson-Crick complement of the first unpaired base of the DNA fragment is detected optically. However, the signal generated by sequencing a single DNA molecule is weak, and thus its detection requires complex and expensive hardware. Ensemble-based systems provide an efficient alternative: they amplify the signal by sequencing a large number of identical copies of the DNA fragment in parallel. To fully reap the benefits of having multiple signal sources, extension of complementary strands should progress at the same rate (so that the signals add in phase). However, synthesis of strands in an ensemble gets out-of-sync due to an occasional failure of nucleotide incorporation in some strands, and premature extension of others. These so-called phasing effects, probabilistic in nature, limit the achievable accuracy and read-lengths of sequencing-by-synthesis. The goal of the proposed project is to develop practical algorithms for optimal base-calling in sequencing-by-synthesis systems, improving their effective read-lengths and accuracy. To this end, we rely on concepts and tools from signal processing and information theory. We address two broadly employed systems: Illumina's four-color platform and Roche's (454 Life Sciences) pyrosequencing platform. If successful, as we expect based on preliminary results, our research will have immediate impact on various applications which require high-performance DNA sequencing.