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Our analysis demonstrated that most genes in the D. melanogaster genome contain 2 or more (distinct) sequences that show sequence similarity (containing 3 or less mismatches) to the same off-target gene.
The number of clusters with 3 or more distinct sequences was negligible at all thresholds, and therefore the number of both homozygous and heterozygous clusters converged to the true value once the clustering threshold was raised to ≥4%.
Only few (2.5%) SAS signals were falsely translated into two or more distinct sequences, and these always included the correct sequence and another sequence being not present in the sample (i.e. false-positive).
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We used all ranks of the NCBI taxonomy, placing more conserved sequences higher up in the taxonomy (i.e., closer to the root) and more distinct sequence onto nodes that are more specific (i.e., closer to the leaves, which represent species and strains).
After removing the proteins that were perfect subsets of other sequences, a total 10,138 unique protein sequences were identified by 3 or more distinct peptide sequences.
Lastly we screened additional siRNAs targeting the IGF-1R specific hits to identify those genes that were targeted by three or more distinct siRNA sequences.
At the same sampling depth (i.e. 500,000 tags) there are many more distinct tag sequences in the LongSAGE library than in the MPSS library (151,794 vs. 12,140).
In our sample, among the sequences with positive Blast hits, 422 were represented by two or more distinct clusters, 64 sequences by 4 or more clusters and 11 genes were represented by 10 to 42 clusters (Table 2).
Proteins were considered present in a given sample, if they had two or more peptides with distinct sequences, having a unique count greater than or equal to one in the respective sample.
The single run produced more than 168,900 distinct sequences with 27,290 sequences having an above cut-off BLAST result.
More than 116,787 distinct sequences were produced with 39,200 sequences below a BLAST cut-off threshold of 10-5.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com