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The sequence alignment of Cluster IV toxins is shown in supplementary figure S3 (Supplementary Material online).
We built PSI-BLAST profiles (position-specific substitution matrix) for each cluster, using the alignment of cluster members as an input against NR database at an Evalue = 10-10.
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The clusters with no hits in the TrED EST sequences and NR were used for the following steps: (1) alignment to non-coding RNA sequences with rRNA sequences downloaded from Rfam and GenBank [ 40], (2) identification of tRNAs with tRNAscan-SE (version 1.1) [ 41], and (3) alignment of clusters to Rfam sequences using HMMER (version 3.0) [ 42] and INFERNAL (version 1.0.2).
MCL was run by setting the inflation parameter (I equal to 4. The alignments of clustered elements were produced by PCMA [ 20] used with default parameters.
The same software (MUSCLE and FastTree) was used to reconstruct approximate maximum likelihood (ML) phylogenetic trees from multiple alignments of Clusters of Orthologous Group COGG) representatives that were used for Xenologous Gene Displacement (XGD) detection.
Quality filtered CCS sequences were clustered based on a 98% similarity threshold with the -cluster_fast option and resulting alignments of clusters containing ≥ 7 sequences (i.e., a 7× threshold) were imported into the Geneious software package (v6.1; http://www.geneious.com) re-aligned using the MAFFT (v7.017) alignment plugin and then manually edited for accuracy.
Alignments of clustered MITE sequences were examined manually, and 66 of these were discarded due to poor overall similarity or due to extensive similarity in the sequences flanking the inverted repeats, indicative of the inverted repeat being within a larger repeat element.
Hammock performs several clustering steps to identify clusters of sequences sharing a motif and generate a multiple sequence alignment of each cluster.
The frequent activities along with their sequential orders are then identified in each cluster, and they are subsequently used as a template to guide the construction of a multiple alignment of the cluster of sequences.
This is most likely to be just a chance alignment of the cluster and the dust clouds," ESO officials wrote in a photo description.
The multiple alignment of each cluster was scanned for positions at which residues in the Xcc and Xoo sequences vs. residues in the Xoc, Xca, Xac, and Xav sequences were more similar within these groups than across them.
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