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All mt gene alignments were subsequently inspected manually.
RRTs were applied to 16 individual mt gene alignments (13 protein-coding genes, 2 rRNA genes, and a single alignment combining all tRNA genes) at the nucleotide level, and neobatrachians had higher mean relative rates in all cases but the nad3 gene (Table 1).
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Phylogenetic analyses were based on the concatenation of the nucleotide sequences of all mt single-gene alignments (hereafter the mt nucleotide data set) or all nuclear single-gene alignments (hereafter the nuclear nucleotide data set), as well as on the same alignments but with open reading frames translated into proteins (hereafter the mt amino acid and the nuclear amino acid data sets).
Ambiguously-aligned positions in both mt trn and rrn gene alignments were excluded using Gblocks as described above.
Ambiguously aligned positions in both mt tRNA and rRNA gene alignments were excluded with Gblocks v.0.19b [ 56] using the following settings: minimum number of sequences for a conserved position 31, minimum number of sequences for a flanking position 36, maximum number of contiguous non-conserved positions 5, minimum length of a block 10, allowed gap positions with half.
Individual mt genes were aligned using the Geneious translational alignment and subsequent manual adjustments, and unalignable codons were identified and removed manually prior to phylogenetic analyses.
We have characterized start and stop codons of S. minutum mt genes using manual alignments between genomic and transcriptomic sequences (supplementary fig. S2, Supplementary Material online).
The reverse primer hybridizes in the second exon of the mt gene, and consequently allows the alignment of the previously described Orchesella cincta mt sequences (Timmermans et al. in press) [ 25]) to the resulting Orchesella cincta pmt sequences.
Duplicated non-coding sequence present coincided with pseudo mt gene in booklice multipartite mt genomes.
It infers that mt genome fragmenting is the important inducement for pseudo mt gene appearance in booklice.
This explanation may be applicable for multipartite mt genome in Liposcelis, pseudo mt gene would be residues of interchromosome recombination.
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