TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets

Junier, Pilar; Junier, Thomas; Witzel, Karl-Paul

doi:10.1128/aem.01394-08

Cited by 84 publications

(75 citation statements)

References 12 publications

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“…PCR products were purified from the reaction mix using magnetic beads (Agincourt, Beverly, MA, USA). Selection of restriction enzymes for each of the genes was based on in silico analysis of clone library sequences (from this study) using the ARB (Ludwig et al, 2004) and TRiFLe packages (Junier et al, 2008). Fluorescently labelled PCR products (200 ng) were digested with HhaI and HpyCH4V (New England Biolabs, Ipswich, MA, USA), for nirS and AOA, respectively.…”

Section: T-rflp Analysis Of Nirs and Aoamentioning

confidence: 99%

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

et al. 2009

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Nitrification and denitrification are key steps in nitrogen (N) cycling. The coupling of these processes, which affects the flow of N in ecosystems, requires close interaction of nitrifying and denitrifying microorganisms, both spatially and temporally. The diversity, temporal and spatial variations in the microbial communities affecting these processes was examined, in relation to N cycling, across 12 sites in the Fitzroy river estuary, which is a turbid subtropical estuary in central Queensland. The estuary is a major source of nutrients discharged to the Great Barrier Reef nearshore zone. Measurement of nitrogen fluxes showed an active denitrifying community during all sampling months. Archaeal ammonia monooxygenase (amoA of AOA, functional marker for nitrification) was significantly more abundant than Betaproteobacterial (b-AOB) amoA. Nitrite reductase genes, functional markers for denitrification, were dominated by nirS and not nirK types at all sites during the year. AOA communities were dominated by the soil/sediment cluster of Crenarchaeota, with sequences found in estuarine sediment, marine and terrestrial environments, whereas nirS sequences were significantly more diverse (where operational taxonomic units were defined at both the threshold of 5% and 15% sequence similarity) and were closely related to sequences originating from estuarine sediments. Terminal-restriction fragment length polymorphism (T-RFLP) analysis revealed that AOA population compositions varied spatially along the estuary, whereas nirS populations changed temporally. Statistical analysis of individual T-RF dominance suggested that salinity and C:N were associated with the community succession of AOA, whereas the nirS-type denitrifier communities were related to salinity and chlorophyll-a in the Fitzroy river estuary.

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Section: T-rflp Analysis Of Nirs and Aoamentioning

confidence: 99%

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

et al. 2009

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“…Data analysis was carried out as described in Junier et al (2008c). Simulations of terminal restriction fragments (T-RFs) from clonal sequences from this study were carried out using TRiFLe (Junier et al 2008a). Statistical analyses were carried out using the softwares Primer E (clustering and multidimensional scaling; Clarke 1993) and R (www.R-project.org).…”

Section: Methodsmentioning

confidence: 99%

Methane- and ammonia-oxidizing bacteria at the chemocline of Lake Kinneret (Israel)

Junier¹,

Kim²,

Eckert³

et al. 2010

Aquat. Microb. Ecol.

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The vertical distribution of methane- and ammonia-oxidizing bacteria (MOB and AOB, respectively), and the physicochemical conditions in the chemocline of Lake Kinneret (Israel) were studied at a resolution of 10 cm from 16.2 to 17.7 m depth. Profiles of the chemical parameters indicated decreasing concentrations of methane (from 22.4 to 0.11 µmol l–1) and ammonia (from 14.2 to 8.4 µmol l–1) towards the water surface and in close proximity to the chemocline. The disappearance of methane coincided with methane oxidation that could be corroborated throughout this layer with highest rates at 17.4 to 17.6 m. Disappearance of ammonia could not be linked to ammonia oxidation exclusively. The genes pmoA and the homologous amoA (coding for subunit α of the methane and ammonia monooxygenase, respectively) were amplified by PCR. The products were analyzed by terminal restriction fragment length polymorphism (T-RFLP) and sequencing of clone libraries. The results demonstrated that different MOB and AOB communities are established along the concentration gradient within the narrow layer of the metalimnetic chemocline. Changes in the intensity of the T-RFLP peaks and the frequency of different groups of alpha- and gammaproteobacterial MOB, and betaproteobacterial AOB, coincided with the concentration gradients of methane, ammonia, nitrate, and oxygen in the chemocline. This suggests that different communities of MOB, and to a lesser extent AOB, contribute to the formation of chemical gradients of their particular substrates in the chemocline

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“…T-Align generate a consensus profile from replicate data and compare generated consensus profile to get the result about community structure and composition, while PAT provide phylogenetic assignment in terms of species diversity and composition from user supplied data. TRF-CUT and TRiFLe introduced by Ricke et al [45] and Junier et al [48] respectively generate theoretical T-RFs by in silico digestion of user supplied database of small subunit rRNA gene sequences or any other functional gene of interest. In addition Stres et al [49] developed BEsTRF to get the optimal resolution of T-RFLP data by using the user defined primer-enzymesequence database.…”

Section: Tools For Data Analysismentioning

confidence: 99%

“…uidaho.edu/) and T-RFPred (http://nodens.ceab.csic.es/trfpred/) are available for the in silico digestion of 16S rRNA (database) sequences with a number of restriction enzymes and prediction of their relative T-RFs' patterns. Recently, a number of applications like phylogenetic assignment tool (PAT) [47], T-Align (http://inismor.ucd.ie/ *talign/ [43]), ARB software integrated tool, TRF-CUT (http://www.mpi-marburg.mpg.de/braker/trfcut.zip [47]) TRiFLe (http://cegg.unige.ch/trifle/trifle.jnlp [48]) and T-RFLP statistical data analysis software (http://www. ibest.uidaho.edu/tool/T-RFLP_stats/index.php) [1] are available to perform tasks like profile comparison, statistical analysis of data and representation of similarity in the form of a dendrogram.…”

Section: Tools For Data Analysismentioning

confidence: 99%

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

et al. 2014

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Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.

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TRiFLe, a Program for In Silico Terminal Restriction Fragment Length Polymorphism Analysis with User-Defined Sequence Sets

Cited by 84 publications

References 12 publications

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary

Methane- and ammonia-oxidizing bacteria at the chemocline of Lake Kinneret (Israel)

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

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