Sequencing Depth
For EvercodeTM WT FFPE libraries, we recommend a minimum sequencing depth of 20,000 reads per nucleus. Ideal sequencing depth is dependent on your experimental goals. The flexibility of sublibrary creation in the Parse Biosciences workflow allows you to start with sequencing one sublibrary more deeply to assess library complexity and sequencing saturation. In turn, this can inform how to optimize sequencing depth for the rest of the sublibraries in the experiment.
Run configuration
Sequencing libraries generated with Evercode WT FFPE kits require 100 cycle kits for Illumina sequencing. Note that 100 cycle kits actually include reagents for 138 total cycles.
Details of the final sequencing library structure are below.
Libraries should be sequenced with paired reads using the read structure in the table below. Read lengths longer than those recommended below are acceptable, but additional bases in read 2 are trimmed by the Parse Analysis Pipeline.
| Read | Function | Cycles |
| Read 1 | cDNA Insert | 64 |
| i7 Index (Index 1) | Barcode 4 / UDI | 8 |
| i5 Index (Index 2) | Barcode 4 / UDI | 8 |
| Read 2 | Barcodes 1-3 | 58 |
Sequencing libraries should be diluted and denatured according to the manufacturer’s instructions for the relevant sequencing instrument. We strongly recommend adding 5% PhiX for optimal sequencing quality.