Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.
Sliding window analyses for optimal selection of mini-barcodes, and application to 454-pyrosequencing for specimen identification from degraded DNA.
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DOI：
10.1371/journal.pone.0038215
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Wratten SD
中科院分区：
文献类型：
--
作者：
Boyer S;Brown SD;Collins RA;Cruickshank RH;Lefort MC;Malumbres-Olarte J;Wratten SD
当应用于饮食分析、古代 DNA 研究、环境 DNA 样本以及更广泛的 DNA 样本未得到充分保存的任何情况下，DNA 条形码仍然是一个挑战。由于常用的条形码标记 (COI) 的大小超过 600 个碱基对 (bp)，因此当 DNA 分子降解为更小的片段时，扩增会失败。然而，用于标本识别的相关信息可能不会沿着条形码区域均匀分布，并且较短的目标足以用于识别目的。这项研究提出了一种新的、广泛适用的方法来比较给定分子标记的所有潜在“迷你条形码”的性能，并客观地选择最短和信息最多的一个。我们的方法基于新的 R 包 SPIDER（R 中的物种识别和进化）中实现的滑动窗口分析。该方法适用于任何分类单元和任何分子标记。在这里，它对通过肉食性蜗牛消化而降解的蚯蚓 DNA 进行了测试。选择 16 S rDNA 的 100 bp 区域作为准确标本识别所需的最短信息片段（迷你条形码）。设计了相应的引物，并使用 454 焦磷酸测序从 46 个蜗牛（捕食者）粪便中扩增降解的蚯蚓（猎物）DNA。这导致在蜗牛的饮食中检测到 18 种蚯蚓。我们鼓励分子生态学家使用这种方法客观地选择他们想要从降解的 DNA 中扩增的基因中信息最丰富的区域。这里提供的方法和工具特别有用：(1) 在处理只能扩增小片段的降解 DNA 时，(2) 对于尚未就适当的条形码基因达成共识的情况，或 (3) 允许直接分析来自大规模并行测序的短读数，而不需要生物信息学整合。
DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation.
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