Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis

Wu, Liyou; Wen, Chongqing; Qin, Yujia; Yin, Huaqun; Tu, Qichao; Nostrand, Joy D. Van; Yuan, Tong; Yuan, Menting; Deng, Ye; Zhou, Jizhong

doi:10.1186/s12866-015-0450-4

Cited by 211 publications

(176 citation statements)

References 43 publications

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“…Although not currently in widespread use, this approach does appear to ameliorate the problems caused by low sequence diversity, in some cases improving sequence base read quality by 10%, raw sequence throughput by 15% and the number of effective reads by nearly 50% (Wu et al 2015). Illumina MiSeq platforms are commonly used for amplicon DNA sequencing and a wide range of instrument and sequencing chemistry options are available from Illumina.…”

Section: Illuminamentioning

confidence: 99%

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Lear¹,

Dickie²,

Banks³

et al. 2018

100

112

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Advances in the sequencing of DNA extracted from media such as soil and water offer huge opportunities for biodiversity monitoring and assessment, particularly where the collection or identification of whole organisms is impractical. However, there are myriad methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. To help overcome potential biases that may impede the effective comparison of biodiversity data collected by different researchers, we propose a standardised set of procedures for use on different taxa and sample media, largely based on recent trends in their use. Our recommendations describe important steps for sample pre-processing and include the use of (a) Qiagen DNeasy PowerSoil ® and PowerMax ® kits for extraction of DNA from soil, sediment, faeces and leaf litter; (b) DNeasy PowerSoil ® for extraction of DNA from plant tissue; (c) DNeasy Blood and Tissue kits for extraction of DNA from animal tissue; (d) DNeasy Blood and Tissue kits for extraction of DNA from macroorganisms in water and ice; and (e) DNeasy PowerWater ® kits for extraction of DNA from microorganisms in water and ice. Based on key parameters, including the specificity and inclusivity of the primers for the target sequence, we recommend the use of the following primer pairs to amplify DNA for analysis by Illumina MiSeq DNA sequencing: (a) 515f and 806RB to target bacterial 16S rRNA genes (including regions V3 and V4); (b) #3 and #5RC to target eukaryote 18S rRNA genes (including regions V7 and V8); (c) #3 and #5RC are also recommended for the routine analysis of protist community DNA; (d) ITS6F and ITS7R to target the chromistan ITS1 internal transcribed spacer region; (e) S2F and S3R to target the ITS2 internal transcribed spacer in terrestrial plants; (f) fITS7 or gITS7, and ITS4 to target the fungal ITS2 region; (g) NS31 and AML2 to target glomeromycota 18S rRNA genes; and (h) mICOIintF and jgHCO2198 to target cytochrome c oxidase subunit I (COI) genes in animals. More research is currently required to confirm primers suitable for the selective amplification of DNA from specific vertebrate taxa such as fish. Combined, these recommendations represent a framework for efficient, comprehensive and robust DNA-based investigations of biodiversity, applicable to most taxa and ecosystems. The adoption of standardised protocols for biodiversity assessment and monitoring using DNA extracted from environmental samples will enable more informative comparisons among datasets, generating significant benefits for ecological science and biosecurity applications.

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Section: Illuminamentioning

confidence: 99%

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Lear¹,

Dickie²,

Banks³

et al. 2018

100

112

View full text Add to dashboard Cite

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“…Taxonomy was assigned using the Greengene database (http://greengenes.secondgenome.com/) with PyNAST at 80% confidence threshold. Shannon diversity index (H), observed species, and species equitability were generated for each sample using QIIME .…”

Section: Methodsmentioning

confidence: 99%

Osmotic Pressure Compensated Solute Ectoine Improves Salt Tolerance of Microbial Cells in Microbial Fuel Cells

et al. 2019

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In the technology of microbial fuel cells (MFCs) for simultaneous electricity production and wastewater purification, the electricity production of MFCs decreases when the salt concentration in the wastewater is high. Increasing the electricity production of MFCs under high salt conditions is a great challenge. This paper studies the reasons for the decline of electricity production of MFCs under high salt conditions and develops ways to increase the electricity generation. The results show that high salt inhibits the growth of electrogenic microorganisms on the anode of MFCs, thereby reducing the electricity production. Addition of ectoine, an osmotic pressure compensated solute, can increase the salt tolerance of electrogenic microorganisms, thereby increasing the electricity generation of MFCs. With the addition of 1 mmol L−1 ectoine to the substrate, the average voltage output of the MFCs based on mixed microbial flora anode was increased by 60.4% (±3.9%). Under this condition, microbial diversity analysis showed that the dominant growth and electricity production genus Acinetobacter accounted for 38.9% in the microbial community of anode biofilm.

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“…Illumina MiSeq is performed on a bench‐top device and is designed for speed and simplicity for targeted resequencing, for example PCR amplicons and small‐genome sequencing such as microorganisms, mitochondrial, and chloroplast genomes. Illumina MiSeq amplicon sequencing is a dominant platform in the study of microbial ecology and genotyping of disease related genes …”

Section: Introductionmentioning

confidence: 99%

Investigating target‐site resistance mechanism to the PPO‐inhibiting herbicide fomesafen in waterhemp and interspecific hybridization of Amaranthus species using next generation sequencing

Nie

Mansfield

Harre

et al. 2019

Pest Management Science

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BACKGROUND Waterhemp (Amaranthus tuberculatus (Moq.) J. D. Sauer) is one of the most pernicious weeds in cropping systems of the USA due to its evolved resistance against several herbicide sites‐of‐action, including protoporphyrinogen oxidase inhibitors (PPO‐R). Currently, the only source of PPO‐R documented in waterhemp is ΔG210 of PPX2. Gene flow may not only lead to a transfer of herbicide‐resistant alleles, but also produce a hybrid genotype more competitively fit than one or both parents. However, investigating gene flow of Amaranthus species has been of interest in the past two decades with limited evidence. RESULTS Here, a high‐throughput MiSeq amplicon sequencing method was used to investigate alterations of the PPX2 gene in 146 PPO‐R waterhemp populations across five Midwest states of the USA. Five R128 codons of PPX2, novel to waterhemp, were found including AGG (R), GGA (G), GGG (G), AAA (K) and ATA (I). R128G, R128I, and R128K were found in 11, 3, and 2 populations, respectively. R128G and R128I, but not R128K, conferred fomesafen resistance in a bacterial system. Sequence alignment of the R128 region of PPX2 identified a tumble pigweed (Amaranthus albus)‐type and Palmer amaranth (Amaranthus palmeri)‐type PPX2 allele to be present and widespread in the surveyed waterhemp populations, thus providing strong evidence of gene flow between Amaranthus species. CONCLUSION Using a next‐generation sequencing method, we identified two PPO target‐site mutations R128G/I novel to waterhemp and provided evidence of gene flow of Amaranthus species in a large group of screened waterhemp populations from five Midwest states of the USA. © 2019 Society of Chemical Industry

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Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis

Cited by 211 publications

References 43 publications

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

Osmotic Pressure Compensated Solute Ectoine Improves Salt Tolerance of Microbial Cells in Microbial Fuel Cells

Investigating target‐site resistance mechanism to the PPO‐inhibiting herbicide fomesafen in waterhemp and interspecific hybridization of Amaranthus species using next generation sequencing

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