MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

Background
Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.
Methods
To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.
Results
The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.
Conclusions
Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.

背景
焦磷酸测序（pyrosequencing）可用于基于单核苷酸多态性（Single-Nucleotide-Polymorphism, SNP）的病原体分型，或获取短DNA片段的序列信息。然而，部分病原体的分子分型无法仅依靠单个SNP或短序列片段完成，因此需纳入多个基因组区域进行考量。一种颇具前景的快速方法是同时使用多条测序引物，即多重焦磷酸测序（multiplex pyrosequencing）。此类引物可生成指纹型焦磷酸图谱，该图谱由所有所用引物各自产生的焦磷酸图谱叠加构成。
方法
为优化基于焦磷酸测序的病原体分型技术，我们开发了软件工具MultiPSQ，可加速多重焦磷酸图谱的分析与评估工作。作为概念验证，我们开发了一种用于正痘病毒（orthopoxviruses）分型的多重焦磷酸测序检测方法，用于分析德国痘病毒咨询实验室诊断的临床样本。
结果
软件工具MultiPSQ可对不同测序引物产生的多重焦磷酸图谱进行分析。因此，基于焦磷酸测序的病原体分型可使用多个靶标区域。如概念验证检测实验所示，MultiPSQ可通过两条引物准确识别不同正痘病毒毒株中存在的SNP。
结论
目前已有的软件仅支持固定数量的SNP与测序引物，极大限制了该技术的应用价值。与之相比，我们研发的新型软件MultiPSQ可分析包含任意数量测序引物、覆盖任意数量多态性位点的多重焦磷酸测序检测数据。