Department of Molecular Ecology, Radboud University Nijmegen, Institute for Water and Wetland Research, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands.
Mol Ecol Resour. 2011 Jul;11(4):662-74. doi: 10.1111/j.1755-0998.2011.02990.x. Epub 2011 Feb 21.
Next-generation sequencing (NGS) technologies are increasingly applied in many organisms, including nonmodel organisms that are important for ecological and conservation purposes. Illumina and 454 sequencing are among the most used NGS technologies and have been shown to produce optimal results at reasonable costs when used together. Here, we describe the combined application of these two NGS technologies to characterize the transcriptome of a plant species of ecological and conservation relevance for which no genomic resource is available, Scabiosa columbaria. We obtained 528,557 reads from a 454 GS-FLX run and a total of 28,993,627 reads from two lanes of an Illumina GAII single run. After read trimming, the de novo assembly of both types of reads produced 109,630 contigs. Both the contigs and the >75 bp remaining singletons were blasted against the Uniprot/Swissprot database, resulting in 29,676 and 10,515 significant hits, respectively. Based on sequence similarity with known gene products, these sequences represent at least 12,516 unique genes, most of which are well covered by contig sequences. In addition, we identified 4320 microsatellite loci, of which 856 had flanking sequences suitable for PCR primer design. We also identified 75,054 putative SNPs. This annotated sequence collection and the relative molecular markers represent a main genomic resource for S. columbaria which should contribute to future research in conservation and population biology studies. Our results demonstrate the utility of NGS technologies as starting point for the development of genomic tools in nonmodel but ecologically important species.
下一代测序（NGS）技术在许多生物体中得到了越来越多的应用，包括对生态和保护目的很重要的非模式生物。Illumina 和 454 测序是应用最广泛的 NGS 技术之一，当它们一起使用时，能够以合理的成本产生最佳的结果。在这里，我们描述了这两种 NGS 技术的联合应用，以描述一种生态和保护相关的植物物种的转录组特征，该物种没有可用的基因组资源，即山萝卜（Scabiosa columbaria）。我们从 454 GS-FLX 运行中获得了 528,557 个读数，从两个 Illumina GAII 单运行的通道中总共获得了 28,993,627 个读数。在读取修剪后，两种类型的读取的从头组装产生了 109,630 个 contigs。将 contigs 和 >75 bp 的剩余单序列分别与 Uniprot/Swissprot 数据库进行 Blast，分别得到 29,676 和 10,515 个显著匹配。基于与已知基因产物的序列相似性，这些序列代表至少 12,516 个独特的基因，其中大多数都被 contig 序列很好地覆盖。此外，我们还鉴定了 4320 个微卫星位点，其中 856 个位点具有适合 PCR 引物设计的侧翼序列。我们还鉴定了 75,054 个潜在的 SNPs。这个注释序列集和相对分子标记代表了山萝卜的主要基因组资源，这应该有助于未来在保护和种群生物学研究中的应用。我们的结果表明，NGS 技术可以作为开发非模式但具有生态重要性的物种基因组工具的起点。
