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Genome supercontigs containing the RFLP marker sequences were downloaded from VectorBase and screened with Tandem Repeats Finder (TRF) software using the default parameters [25].
Each of the 148 DArT marker sequences were queried against 1) all other sequenced DArT markers in the same study and 2) GenBank (www.ncbi.nlm.nih.gov) to identify putative sequence identities using the Basic Local Alignment Search Tool [23] for nucleotides (blastn) and translated to proteins (tblastx).
Orthologs for a majority of Ambystoma marker sequences were identified in more than one reference genome.
Here, C3 marker sequences were defined as the sequences that all C3 species were consistent (without considering F. pringlei), and C3-C4 marker sequences were defined as those sequences that all C3-C4 species were consistent (without considering F. angustifolia).
Inversions of marker sequences were filtered out by discarding inconsistent loci with the exception of very closely linked markers.
The ordered marker sequences were confirmed by the 'Ripple' command and finally the linkage groups were generated by 'Map' command.
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Further, marker sequences are not randomly dispersed, especially for sequence repeats such as SSRs.
In meiotic linkage mapping, relationships among polymorphic marker sequences are determined by relative frequency of recombination.
Primer pairs targeting the SCAR marker sequence were designed, which enabled species-specific detection and avoided cross-amplification of other species of Serranidae fish.
If none of the results returned for a marker sequence were supported, then the top hit was retained, despite the lack of support.
Together the MATα locus and LEU2 marker sequence were amplified using PCR and transformed into YPH278 to replace the SUP11 and URA3 sequence on the present mini-chromosome (MC).
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