Gardner Shea N, Jaing Crystal J, Elsheikh Maher M, Peña José, Hysom David A, Borucki Monica K
Computations, Lawrence Livermore National Laboratory (LLNL), Livermore, CA 94550, USA.
Physical and Life Sciences/Global Security, Lawrence Livermore National Laboratory (LLNL), Livermore, CA 94550, USA.
Adv Bioinformatics. 2014;2014:101894. doi: 10.1155/2014/101894. Epub 2014 Aug 3.
Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.
背景。靶向富集可提高复杂样本中低浓度高变异性病毒的覆盖度。与保守区域退火的简并引物能够促进对不同的低浓度变异体进行扩增，即便所存在的毒株未知。结果。本文描述了一种用于设计多重简并测序引物组的工具，该引物组可跨多个全基因组平铺重叠扩增子。新脚本run_tiled_primers是PriMux软件的一部分。针对南美出血热病毒、蜱传脑炎病毒、亨尼帕病毒、沙粒病毒、丝状病毒、克里米亚-刚果出血热病毒、裂谷热病毒和日本脑炎病毒的每个片段设计了引物。每组病毒的差异都很大，基因组一致性低至5%。针对NCBI核苷酸序列数据库对引物组进行了非靶标交叉反应的计算检查。在实验室中证实，鼠肝炎病毒的引物可特异性扩增来自一株在体外和体内经过多次传代培养的实验室毒株的选定基因。结论。该软件应有助于研究人员在测序前设计用于靶向全基因组富集的多重引物组，以更好地覆盖低滴度、不同的病毒。其应用包括从复杂背景中发现病毒，以及提高基因家族中快速进化的毒株或变异体的灵敏度和覆盖度。
