454 pyrosequencing to describe microbial eukaryotic community composition, diversity and relative abundance: a test for marine haptophytes.
454 pyrosequencing to describe microbial eukaryotic community composition, diversity and relative abundance: a test for marine haptophytes.
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DOI：
10.1371/journal.pone.0074371
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Edvardsen B
中科院分区：
文献类型：
--
作者：
Egge E;Bittner L;Andersen T;Audic S;de Vargas C;Edvardsen B
核糖体DNA的下一代测序越来越多地用于评估微生物群落的多样性和结构。在这里，我们测试454焦磷酸测序的能力，以检测存在的物种数量，并根据细胞数量和原生生物的生物量评估Haptophyta门中原生生物的相对丰度。我们使用了一个由11种触生生物相同数量的细胞组成的模拟群落，并比较了靶向DNA和RNA/cDNA，以及两对不同的V4 SSU rDNA触生生物偏向的引物对。此外，我们测试了四种不同的生物信息过滤方法，以减少结果序列数据集中的错误。测序深度为11000-20000个阅读数，以触角吸虫特异性引物Hap454为靶标，检测到全部11个种。对作为采样深度函数的预期恢复物种数量的稀疏性分析表明，这里需要至少1400个读数才能恢复模拟群落中的所有物种。相对读数丰度与相对细胞数无相关性。虽然生物量最大的物种在阅读中也是比例最丰富的，但以DNA和cDNA为模板，不同物种的比例阅读丰度和比例生物量之间普遍存在较弱的相关性。454测序产生了相当大的假多样性，并且以cDNAs为模板比以dna为模板的多。在只基于条形码和引物匹配的初始筛选中，我们观察到99%相似性的可操作分类单位(OTU)比模拟群落中存在的物种数量多100倍。根据质量分数进行过滤，或者用PyroNoise去噪，产生的OTU99%是物种数量的10倍。使用AmplicNoise降噪后，OTU99%的数量减少到与模拟群落中存在的物种数量相匹配。基于我们的分析，我们提出了一种使用454焦磷酸测序来更准确地描述触须细胞多样性的策略。
Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing.
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影响因子：
4.9
作者：
Bittner, Lucie;Gobet, Angelique;De Vargas, Colomban
通讯作者：
De Vargas, Colomban
影响因子：
1.6
作者：
Edvardsen, B;Medlin, L
通讯作者：
Medlin, L
DOI：
10.1073/pnas.1001665107
发表时间：
2010-08-17
影响因子：
11.1
作者：
Cuvelier, Marie L.;Allen, Andrew E.;Worden, Alexandra Z.
通讯作者：
Worden, Alexandra Z.
影响因子：
3.9
作者：
Evans, J;Sheneman, L;Foster, J
通讯作者：
Foster, J
影响因子：
2.5
作者：
DORTCH, Q;ROBERTS, TL;AHMED, SI
通讯作者：
AHMED, SI