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AS events were predicted based on EST discrepancies only, not on discrepancies between ESTs and annotations.
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ESTs showing discrepancies for location among three homoeologous chromosomes were not considered for analysis.
As ESTs have frequent sequencing errors, only positions with a quality score above 39 were considered, and sequence discrepancies between ESTs in the same contig were marked as putative SNPs only if the polymorphism was confirmed by more than one EST in the contig.
Other discrepancies in EST-SSRs motif abundance and distribution across different plant studies can be attributed to the parameter setup [ 25], annotation deficiency [ 44], and the selected EST-SSR analysis algorithm [ 20].
These three observations: 1) the presence of a predicted signal peptide, 2) the discrepancy in EST abundance between wing and accessory gland tissue, and 3) the tissue-specific patterns of expression, are consistent with these transcripts being ACP genes and suggest they encode seminal fluid proteins.
The reason for this discrepancy between EST and proteomic findings has not been adequately addressed but one explanation could be related to the occurrence of IR in these genes that may interfere with adequate gene transcription and/or translation in vivo, thereby preventing toxin production.
It is possible that some of the discrepancies between the ESTs we assembled and Sanger ESTs or known proteins are true differences, for example, paralogous transcripts or alternatively spliced isoforms.
In the algorithm implemented by MIRA the decision on whether discrepancies between similar EST sequences are significant or not relies more on the underlying quality information data.
Discrepancies between individual ESTs were resolved to maximize sequence similarity to highly similar homologs.
QualitySNP first identified haplotypes in a contig by re-clustering its ESTs and extracted all nucleotide discrepancies (called potential SNPs, pSNPs) between identified haplotypes in a contig, from which a subset of so-called quality SNPs (qSNPs) was identified based on allele and SNP confidence scores defined in the haplotype-based mining algorithm [ 8].
Information from EST sequences was used when any discrepancy was found.
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