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Assembly by read mapping was reliable enough to uncover a single nucleotide error in the reference sequence.
A nucleotide error might not necessarily change the amino acid of the respective codon and thus the prediction score.
The nucleotide error rate and heterozygosity present in the animals sequenced, taken together, are insufficient to explain the high number of sequence-different paralogues we found.
Because the MIDs are designed with a difference of at least four nucleotides between each other, it is possible to easily "correct" up to one nucleotide error in the barcode (TB-0905 Roche MID Adaptors) without a decrease in accuracy.
Notably, in all scenarios where quasispecies theory could potentially apply, i.e. stem and progenitor cells proliferation and cancer cells, aneuploidy rates far exceed single nucleotide error rates in frequency and impact on the cell.
Although such a database can be constructed via NGS, the reads generated by NGS are error-prone and even a single nucleotide error precludes identification of a peptide by the standard proteomics tools.
Similar(47)
The 99th percentile of mismatches would establish the threshold for nucleotide errors in 0.61% and the limit for identifying true drug resistance mutations in 0.20%.
A second caution about comparing values of ⟨ν⟩∞ from different studies is that its value depends on the nucleotide error-spectrum of the experiment, as different mutagenesis methods create different distributions of nucleotide and amino acid mutation types.
Sequencing the unselected populations at the end of the experiment indicated that this protocol introduced an average of 1.4 ± 0.2 nucleotide mutations, with the nucleotide error-spectrum shown in Table 1.
We found that allowing two nucleotide errors in barcodes resulted in large poor-quality data outputs.
The vectors were then sequenced to confirm that there were no nucleotide errors.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com