Varliero Gilda, Wray Jared, Malandain Cédric, Barker Gary
School of Biological Sciences, University of Bristol, Bristol, UK.
Environmental Expertise, HYDREKA, Lyon, France.
PeerJ. 2021 Apr 29;9:e11120. doi: 10.7717/peerj.11120. eCollection 2021.
Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user's input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree non-selected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and species-specific primers for the detection of species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer.
许多环境和生物医学的生物监测与检测研究旨在探索微生物组样本中特定生物体或基因功能的存在情况。在这种情况下，当通过探索少数基因就能回答研究假设时，靶向PCR方法是合适的。然而，由于环境微生物群落的复杂性，设计特异性引物具有挑战性，并且可能导致非特异性结果。我们设计了PhyloPrimer，这是首个用户友好型平台，可半自动化设计针对感兴趣基因的分类群特异性寡核苷酸（即PCR引物）。PhyloPrimer的主要优势在于能够检索并比对与用户输入匹配的GenBank基因序列，并通过在线动态树探索它们之间的关系。然后，PhyloPrimer会针对从树中选择的基因序列设计特异性寡核苷酸，并利用未从树中选择的序列来寻找并最大化靶向序列与非靶向序列之间的寡核苷酸差异，从而提高寡核苷酸的分类群特异性（阳性/阴性一致方法）。然后，使用最近邻（NN）计算检查设计的寡核苷酸是否存在二级结构，并通过PCR测试检查是否存在脱靶匹配，同时也处理具有简并碱基的寡核苷酸。虽然PhyloPrimer的主要功能是设计分类群特异性寡核苷酸（低至物种水平），但该软件也可用于设计无任何分类特异性的靶向基因的寡核苷酸、从预选序列设计寡核苷酸以及检查预先设计的寡核苷酸。我们使用PhyloPrimer为四个市售的微生物模拟群落设计属特异性和种特异性引物，以检测模拟群落中的物种，从而验证了该流程。该软件在这些模拟微生物群落上表现良好，可在https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer上找到。
