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Each dataset was aligned using the CLUSTALW program version 1.83 [ 65], and inspected for alignment accuracy.
Summed dynamic PET images were inspected for alignment with the corresponding attenuation correction CT images, and manually realigned and re-reconstructed as necessary.
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We aligned the amino acid sequences of each pair of orthologs from nine- and three-spined sticklebacks using MUSCLE [ 77] with default settings and manually inspected for possible alignment artifacts.
The training data was manually constructed and inspected for its alignment against highly conserved protein sequences using the AAT package [ 68] and PASA [ 63] to align the ESTs to the genomic sequence with a stringent criteria of 95% identity over 90% length using gmap [ 69].
These were manually inspected for splice site, alignment and conservation validity, resulting in 40 regions comprising 41 NIPs.
Multiple sequence alignments were obtained using 'Dialign' program [ 33] and inspected manually for alignment artefacts.
Amino acid sequences were aligned by MAFFT http://mafft.cbrc.jp/alignment/server/index.html and each visually inspected for regions of high quality alignment.
FAR and ACCase amino acid sequences were aligned by MAFFT http://align.bmr.kyushu-u.ac.jp/mafft/software/ and each visually inspected for regions of high quality alignment.
In addition, the NA sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (NAI) resistance-associated amino acid mutations (R292K and E119V).
Alignments were inspected for inactivating mutations, which were labelled according to their exon (e.g. 9A is the 5'-most mutation in exon 9, etc).
The sequence alignments were inspected for the presence of SNPs.
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