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The BLAST e-value threshold was set at 1e-10, with a minimum alignment length of 100 nucleotides (33 amino acids).
We used the expectation value 1.0 × 10− as a cutoff and maintained at least 75% sequence similarity between the two sequences with a minimum alignment overlap 80%.
All the alignments were carried out with a minimum alignment ratio of 0.93, and the minimum size of alignment of 20.
Thresholds were set to a minimum alignment length of 70 nucleotides, minimum query coverage of 90% and minimum per cent identity of 80%.
Using a stringent cutoff requiring sequence identity of at least 90% and a minimum alignment length of 30 nucleotides, 509 (38.6%) sequences in our library have a hit in the previous library, and 711 (54%) of the sequences in the earlier library have a hit in ours.
A minimum alignment of at least three identical consensus sequences defined an allelic sequence from the pool of sequences.
¥ Lowest common ancestor (LCA) using 1e-5 cutoff, 60% minimum identity, and a minimum alignment lenght cutoff of 15.
A minimum alignment length l A,min of 20 nt is used for finding pairwise alignments.
The matches were filtered with delta-filter allowing only one to one alignments, a minimum identity of 94% and a minimum alignment length of 1,000 bp.
The pepper genome matches were filtered at 98% identity, minimum alignment length of 200 nucleotides (nt), and a maximum of 50 unaligned nt at either end of the unigene.
Blast alignments were then parsed with custom python scripts to extract and count the best miRNA alignment for each read with a minimum read alignment of 18 nucleotides.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com