How deeply you sequence your Immunoverse libraries depends on your application. Deeper sequencing will allow for the identification of more clonotypes; however, there is a plateau where the user will not see many additional clonotypes despite deeper sequencing. This plateau trends with library diversity, which is driven by input amount, input quality, immune cell content and other biological factors.
For dominant clone identification, the user may only need to sequence to 100,000 reads, while diversity or low abundance applications may require more input and deeper sequencing. A recommended starting point is 1.5 M reads with 400 ng of total RNA to capture most of the sample diversity in the sequencing data.
The rarefaction analysis below was derived from Immunoverse TCR libraries generated from varying amounts of high-quality input from a PBL sample and illustrates the relationship between input amounts, sequencing depths and clonotype count for a diverse sample type.
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