Amaral-Zettler Linda A, McCliment Elizabeth A, Ducklow Hugh W, Huse Susan M
The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA, USA.
PLoS One. 2009 Jul 27;4(7):e6372. doi: 10.1371/journal.pone.0006372.
Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes.
METHODOLOGY/PRINCIPAL FINDINGS: We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments.
CONCLUSIONS/SIGNIFICANCE: Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.
从小亚基（SSU）核糖体RNA（rRNA）基因的V6高变区扩增子进行大规模平行焦磷酸测序，常用于评估细菌和古菌群体的多样性和丰富度。焦磷酸测序技术的最新进展使得读长可达240个核苷酸。如今，扩增子焦磷酸测序可应用于SSU rRNA基因更长的可变区，包括真核生物中的V9区。
方法/主要发现：我们提出了一种用于真核生物环境样本V9区扩增子焦磷酸测序的方案，用于生物多样性清查和物种丰富度估计。国际海洋微生物普查（ICoMM）和跨不同水生长期生态研究站点的微生物清查研究（MIRADA-LTERs）项目已经在广泛的海洋和淡水环境中采用该方案对真核生物样本进行标签测序。
结论/意义：对SSU rRNA基因的真核生物V9高变区进行大规模平行焦磷酸测序，为从深度采样群体中估计物种丰富度以及从环境中发现新物种提供了一种方法。
