Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

Eland, Lucy E.; Davenport, Russell J.; Mota, César R.

doi:10.1016/j.watres.2012.07.023

Cited by 53 publications

(35 citation statements)

References 42 publications

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“…For phylogenetic analysis based on the 18S rRNA gene sequences, microalgal DNA was extracted using a DNA extraction kit (DNeasy Blood & Tissue Kit, Qiagen, Netherlands) in order to remove PCR inhibitors from the microalgae [13]. Then, the 18S rRNA gene amplification was carried out using a PCR premix (AccuPower, Bioneer, Korea) and two eukaryotic primers (Ecol-7F, 5 0 -ACCTGGTTGATCCTGCCAG-3 0 and Ecol-1534R, 5 0 -TGATCCTT-CYGCAGGTTCAC-3 0 ).…”

Section: Phylogenetic Analysis Based On 18s Rrna Gene Sequencesmentioning

confidence: 99%

Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry

Lee

Roh

Cho

et al. 2015

Talanta

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Section: Phylogenetic Analysis Based On 18s Rrna Gene Sequencesmentioning

confidence: 99%

Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry

Lee

Roh

Cho

et al. 2015

Talanta

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“…Sequencing offers the rapid detection of algal species without many of the problems associated with microscopy: (i) identification is not limited to those organisms with well-identified morphological markers; (ii) fewer personnel and less time are required (20); and (iii) hundreds of samples can be processed simultaneously by leveraging massively parallel approaches afforded by high-throughput sequencing. Furthermore, examining the community using amplicon sequencing allows us to take an in-depth look at the community structure of the organisms present.…”

mentioning

confidence: 99%

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

Bradley

Pinto

Guest

2016

Appl Environ Microbiol

215

142

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The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3= end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.T he use of high-throughput sequencing technologies (1, 2) has transformed the field of microbial ecology by contributing to a significant body of work that has changed our understanding of microbially diverse populations in a range of ecosystems. This is particularly true for investigations of bacterial and archaeal communities that target the 16S rRNA gene (1, 3-5). However, amplicon sequencing approaches for eukaryotes hav...

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“…Materials and methods [14], and the 18S rRNA gene was amplified using two eukaryotic primers (Ecol-7F, 5 0 -ACC TGG TTG ATC CTG CCA G-3 0 and Ecol-1534R, 5 0 -TGA TCC TTC YGC AGG TTC AC-3 0 ) and PCR pre-mix (AccuPower ® ; Bioneer, Korea) [15]. The PCR program consisted of an initial denaturation at 94 C for 10 min, 30 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 2 min, and extension at 72 C for 3 min, with a final extension at 72 C for 5 min.…”

mentioning

confidence: 99%

Enhanced biomass and lipid production by supplement of myo-inositol with oceanic microalga Dunaliella salina

Cho¹,

Kim²,

Lim³

et al. 2015

Biomass and Bioenergy

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Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

Cited by 53 publications

References 42 publications

Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry

Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

Enhanced biomass and lipid production by supplement of myo-inositol with oceanic microalga Dunaliella salina

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