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The gene prediction pipeline was structured as follows: peptides from diverse angiosperms and ∼260,000 EST assemblies (from ∼2.9 M filtered E. grandis ESTs and ∼2.4 M EST sequences from other closely related ('sister') Eucalyptus species, assembled with PASA) were aligned to the genome and their overlaps used to define putative protein-coding gene loci.
In addition, when enrichment of upstream consensus matches for individual genes is used to define putative cis-regulatory targets at a false discovery rate of 5% (see Methods), a significant number of genes is found only for E2F (Figure 1C).
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Therefore, the closest flanking loci meeting neutral expectations were used to define the regions harbouring putative selected genes; although we expect selected genes to lie closer to the central locus than either flanking loci.
Bulked segregant analysis (BSA) was used to define a subset of putative SNPs within the candidate gene region, which were then used to fine-map GPC-B1.
However the degree of down-regulation was above the logFC of −1.1 used to define our set of putative miR-143 target.
Therefore a cutoff >77 on the EDS-G was used to define values in a putative pathological range and assess clinically relevant levels of excessive exercising.
The default cutoff of −log(P value) ≥2 was used to define a region having undergone a putative selective sweep.
The EXPASY Translate tool (http://www.expasy.ch/tools/dna.html) was used to define the longest ORF for each GST putative transcript.
Immunohistochemistry was used to define the phenotype of neurones expressing Cx45 and determine putative sources of extrinsic innervation.
Having confirmed that AtoC binds in vivo to its a priori target, ChIP experiments were used to define whether AtoC binds to any of the putative targets that emerged from the bioinformatics approach.
We then added to the right and left of each sampled site flanks of length 4 bp sampled uniformly from the corresponding set of flanks of the same putative motif sites that are used to define the PWM.
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