Directed terminal restriction analysis tool (DRAT): an aid to enzyme selection for directed terminal‐restriction fragment length polymorphisms

Roberts, David M.; Schofield, Pietà; Donn, Suzanne; Daniell, Tim J.

doi:10.1111/j.2041-210x.2011.00139.x

Cited by 6 publications

(3 citation statements)

References 24 publications

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“…The 5¢ end of the gene was selected as it contains the most variability (Floyd et al 2002) and been used in nematode sequencing studies (Floyd et al 2002;Griffiths et al 2006). Restriction enzymes were selected using the freeware application DRAT (Roberts et al 2011) (http://www.hutton.ac.uk/drat). Sequence types from the three treatments were trimmed to include the Nem_SSU_F74 and Nem_18S_R primer sites.…”

Section: E S I G N O F D T -R F L P S T R a T E G Ymentioning

confidence: 99%

A novel molecular approach for rapid assessment of soil nematode assemblages – variation, validation and potential applications

et al. 2011

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Summary1. Nematode assemblages are commonly used as an indicator of ecosystem health; however, conventional approaches to assemblage analyses are restricted by time-consuming processing and declining availability of expertise. Molecular methods offer a rapid and cost-effective alternative. 2. We have designed a molecular profiling system, using directed terminal-restriction fragment length polymorphism (dT-RFLP), to characterise nematode assemblages by relative abundance of feeding guilds.3. An arable soil was first characterised by cloning and sequencing of small subunit ribosomal DNA, and an enzyme digest selected to discriminate between feeding guilds. This yielded 14 different terminal-restriction fragments (T-RFs) from the sequence set, assigned to five nematode feeding guilds. 4. Robustness of the dT-RFLP methodology was tested. The greatest amount of variation between replicates occurred at the PCR stage, with little variability between replicate digests from the same PCR product or capillary runs. 5. dT-RFLP revealed changes in assemblage composition owing to organic amendments of dairycattle slurry and municipal green compost. The proportion of microbial feeding nematodes was higher in compost and slurry plots than in the no amendment control in the first sampling after organic amendment. Plant feeding nematodes composed a significantly greater proportion of the control assemblage during the growing season and post-harvest. 6. The increased throughput of molecular analysis compared with microscopy increases the feasibility of studies involving large-scale sampling and makes nematode assemblage analysis more attractive as an indicator of soil health for environmental assessment.

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Section: E S I G N O F D T -R F L P S T R a T E G Ymentioning

confidence: 99%

A novel molecular approach for rapid assessment of soil nematode assemblages – variation, validation and potential applications

et al. 2011

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“…This limitation has been overcome by REPK (Collins & Rocap ) and DRAT (Roberts et al . ) which allow taxonomic rank discrimination and are not limited to ribosomal RNA genes. Additionally, both programmes are able to perform an automated search for sets of REs that provide higher combined genetic resolution than is usually achieved by an individual RE.…”

Section: Introductionmentioning

confidence: 99%

OEZY: Optimising EnZYme selection for best performing terminal restriction fragment length polymorphism analysis using ARB

et al. 2015

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Summary 1.Terminal restriction fragment length polymorphism (TRFLP) analysis remains a useful technique to obtain insights into the genetic diversity of microbial populations. A crucial parameter of the technique is the selection of appropriate restriction endonucleases (REs) to achieve high resolution between the PCR-amplified fragments of a marker gene (usually a ribosomal RNA gene). However, despite the development of several computer-supported programmes to improve the selection of REs for TRFLP analysis, there is still a lack of software that offers both of two aspects: first, availability of a sequence data base from which sequences can easily and without further formatting and ranking be selected for analysis; secondly, selection of sets of REs for highest genetic resolution while providing the possibility to assess and quantify the correlation of the TRFs with the phylogeny of the target group of 16S rRNA sequences. 2. Here, we present a new and freely available software tool which utilises ARB in combination with the SILVA data base of hundreds of thousands of aligned ribosomal RNA genes or user-submitted sequences as basis for the selection of optimal sets of REs of various sizes. Apart from coping with missing sequence information and providing extensive information on the obtained TRF patterns, this new programme for Optimising EnZYme selection for best performing TRFLP analysis using ARB (OEZY) also assesses the level at which the resulting TRF pattern reflects the phylogeny based on the data base gene sequences. 3. Optimising EnZYme is a substantial extension to hitherto available software as it opens the chance to correctly predict the phylogenetic position of yet unknown sequence types. Choosing REs that lead to a high correlation between the resulting TRFs and the phylogeny of the micro-organisms based on the nucleotide sequence of the marker gene makes it likely that the TRFs also fall within the corresponding phylogenetic clade. OEZY therefore provides a diagnostic tool for the analysis of microbial populations.

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“…This produces community ‘fingerprints’ for samples analysed that can be compared in terms of diversity and composition. The use of TRFLPs alone does not produce sequences of the organisms in a community, but if used in conjunction with sequence databases, putative identities may be assigned to TRFs, allowing their identification [13] , [19] .…”

Section: Introductionmentioning

confidence: 99%

What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities

2014

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Arbuscular mycorrhizal (AM) fungi are widely distributed microbes that form obligate symbioses with the majority of terrestrial plants, altering nutrient transfers between soils and plants, thereby profoundly affecting plant growth and ecosystem properties. Molecular methods are commonly used in the study of AM fungal communities. However, the biases associated with PCR amplification of these organisms and their ability to be utilized quantitatively has never been fully tested. We used Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to characterise artificial community templates containing known quantities of defined AM fungal genotypes. This was compared to a parallel in silico analysis that predicted the results of this experiment in the absence of bias. The data suggest that when used quantitatively the TRFLP protocol tested is a powerful, repeatable method for AM fungal community analysis. However, we suggest some limitations to its use for population-level analyses. We found no evidence of PCR bias, supporting the quantitative use of other PCR-based methods for the study of AM fungi such as next generation amplicon sequencing. This finding greatly improves our confidence in methods that quantitatively examine AM fungal communities, providing a greater understanding of the ecology of these important fungi.

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Directed terminal restriction analysis tool (DRAT): an aid to enzyme selection for directed terminal‐restriction fragment length polymorphisms

Cited by 6 publications

References 24 publications

A novel molecular approach for rapid assessment of soil nematode assemblages – variation, validation and potential applications

A novel molecular approach for rapid assessment of soil nematode assemblages – variation, validation and potential applications

OEZY: Optimising EnZYme selection for best performing terminal restriction fragment length polymorphism analysis using ARB

What Goes in Must Come out: Testing for Biases in Molecular Analysis of Arbuscular Mycorrhizal Fungal Communities

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