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I am trying to build my own transcriptome. At the moment I am at the time of library preparation. I am not sure at which sequencing depth I should sequence my libraries.
What is the minimum number of RNA reads per gene I should sequence, assuming I am targeting roughly 1 million genes? Is this minimum number mainly determined by the size of the library and/or sequencing depth I can afford? If so, what is the maximum size of the library?
Not only would the number of transcripts/gene depend on how noisy your RNA-seq experiment is, but also on the abundance of transcripts.
In addition, you might find that certain transcripts have very good TPM but have a high number of copies (per cell) and thus might not be informative.
What you are looking for here is not the minimum but rather a sample/replicate RNA count for each gene.
From the perspective of sequencing, you are looking at between two and three reads per transcript (plus or minus a few bases for the read quality) depending on the actual number of transcript copies and the amount of noise in your experiment. If you have a reference genome, you can get an idea of the number of transcript copies by looking in Ensembl at the GFF file.
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