A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents.
A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents.
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DOI：
10.1186/1742-9994-10-34
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发表时间：
2013
影响因子：
2.8
通讯作者：
Machida RJ
中科院分区：
文献类型：
--
作者：
Leray M;Yang JY;Meyer CP;Mills SC;Agudelo N;Ranwez V;Boehm JT;Machida RJ
基于PCR的同源基因分析已经成为物种检测和鉴定的最有力的方法之一，特别是最近新一代测序平台(NGS)的出现，使得从广泛的环境样本中识别物种组成成为可能。从这些样本中识别物种依赖于将序列与参考条形码进行匹配的能力，以进行分类鉴定。不幸的是，大多数环境样本的研究都以核糖体标记为目标，尽管线粒体细胞色素c氧化酶亚单位I基因(COI)是公共参考文库中迄今为止最广泛可用的序列区域。这在很大程度上是因为现有的通用COI引物针对的是658条码区域，其大小被认为对许多NGS应用来说太大了。此外，众所周知，传统的条形码引物在某些分类群中保守性很差。我们首先在线粒体COI高变区设计了一种新的聚合酶链式反应引物“mlCOIintF”。然后，我们证明了这一新设计的正向引物与“jgHCO2198”反向引物相结合，针对313个碱基的片段，在后生动物多样性中表现良好，比传统用于DNA条码的通用引物集(即LCO1490/HCO2198)具有更高的成功率。最后，我们展示了如何将较短的COI片段与高效的生物信息学管道相结合，通过焦磷酸测序来表征环境样品中的物种多样性。我们研究了三种浮游性和底食性珊瑚礁鱼(科：Apogonidae和Holocentridae)的肠道内容物。在去除可疑的COI序列后，我们从16条鱼的内脏中获得了334个猎物操作分类单位(OTUS)，隶属于14个门。在这些序列中，52.5%与参考条形码匹配(序列相似性为98%)，另外32%可以使用贝叶斯分配被分配到更高的分类水平。使用新设计的mlCOIintF引物和jgHCO2198引物对以313COI片段为靶标的肠道内容物进行分子分析，为后生动物后生动物的后生动物编码研究提供了巨大的希望。我们相信，对于从大规模生物多样性评估到食物网络研究的一系列应用来说，这套引物将是一笔宝贵的财富。
The PCR-based analysis of homologous genes has become one of the most powerful approaches for species detection and identification, particularly with the recent availability of Next Generation Sequencing platforms (NGS) making it possible to identify species composition from a broad range of environmental samples. Identifying species from these samples relies on the ability to match sequences with reference barcodes for taxonomic identification. Unfortunately, most studies of environmental samples have targeted ribosomal markers, despite the fact that the mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most widely available sequence region in public reference libraries. This is largely because the available versatile (“universal”) COI primers target the 658 barcoding region, whose size is considered too large for many NGS applications. Moreover, traditional barcoding primers are known to be poorly conserved across some taxonomic groups. We first design a new PCR primer within the highly variable mitochondrial COI region, the “mlCOIintF” primer. We then show that this newly designed forward primer combined with the “jgHCO2198” reverse primer to target a 313 bp fragment performs well across metazoan diversity, with higher success rates than versatile primer sets traditionally used for DNA barcoding (i.e. LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment coupled with an efficient bioinformatics pipeline can be used to characterize species diversity from environmental samples by pyrosequencing. We examine the gut contents of three species of planktivorous and benthivorous coral reef fish (family: Apogonidae and Holocentridae). After the removal of dubious COI sequences, we obtained a total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla from 16 fish guts. Of these, 52.5% matched a reference barcode (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment. The molecular analysis of gut contents targeting the 313 COI fragment using the newly designed mlCOIintF primer in combination with the jgHCO2198 primer offers enormous promise for metazoan metabarcoding studies. We believe that this primer set will be a valuable asset for a range of applications from large-scale biodiversity assessments to food web studies.
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DOI：
10.1098/rsbl.2003.0025
发表时间：
2003-08-07
影响因子：
4.7
作者：
Hebert, PDN;Ratnasingham, S;deWaard, JR
通讯作者：
deWaard, JR
影响因子：
16.6
作者：
Fonseca, Vera G.;Carvalho, Gary R.;Sung, Way;Johnson, Harriet F.;Power, Deborah M.;Neill, Simon P.;Packer, Margaret;Blaxter, Mark L.;Lambshead, P. John D.;Thomas, W. Kelley;Creer, Simon
通讯作者：
Creer, Simon
影响因子：
3.7
作者：
Kayal M;Vercelloni J;Lison de Loma T;Bosserelle P;Chancerelle Y;Geoffroy S;Stievenart C;Michonneau F;Penin L;Planes S;Adjeroud M
通讯作者：
Adjeroud M
从高度降解的样品中回收的DNA损伤的量化 - 对粪便中DNA的案例研究。
影响因子：
2.8
作者：
Deagle BE;Eveson JP;Jarman SN
通讯作者：
Jarman SN
影响因子：
4.4
作者：
Ficetola GF;Coissac E;Zundel S;Riaz T;Shehzad W;Bessière J;Taberlet P;Pompanon F
通讯作者：
Pompanon F