Twenty thousand transcripts of varying length and random nucleotide composition were computationally generated and scanned for ORFs. The maximum ORF and transcript lengths were plotted and fitted to a logarithmic curve. The shaded regions represent incidences of randomly occurring ORFs at 1, 2, or 3 standard deviations from the mean. The red line indicates the 300 nt ORF threshold that is commonly used to distinguish protein-coding genes in transcript classification pipelines. Therefore, this plot illustrates that for transcripts longer than ∼1000 bp, such a threshold may define transcripts as protein-coding that would be expected to occur by chance. The function y = 91.Ln(x)−330, which approximates random ORF incidence according to transcript length at two standard deviations above the mean (i.e., 95% confidence interval, indicated in green), could be used to discriminate noncoding from protein-coding transcripts in a transcript-length–dependent manner.

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alpha

$808

$100,000.

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beta

$50,000.

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gamma

$36,000.

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delta

$72,000.

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omicron

$20,000.

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