Sequencing Depth

For Evercode^TM WT v2 or Evercode WT v3 libraries, we recommend a minimum sequencing depth of 20,000 reads per cell. Due to improved library efficiency, for Evercode WT v4 libraries we recommend a minimum of 10,000 reads/cell. However, ideal sequencing depth is dependent on the sample type and experimental goals. For example, if trying to capture rare cell types in a heterogeneous population, deeper sequencing is required. Alternatively, if analyzing a homogeneous population, more shallow sequencing may be appropriate.

We have several datasets on the Parse Biosciences website which can serve as a helpful reference for sequencing depth across different sample types.  

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

Evercode WT v3 and Evercode WT v4 libraries require the same read length. This differs from the read length requirement for Evercode WT v2 sequencing libraries. Please refer to the tables below depending on what type of library you are sequencing.

Evercode Whole Transcriptome v3 or v4

Sequencing libraries generated with Evercode WT v3 or Evercode WT v4 kits require 100 cycle kits for Illumina sequencing. Note that 100 cycle kits actually include reagents for 138 total cycles.

Details of the final whole transcriptome 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.

Whole transcriptome 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.

Evercode WT v2

Sequencing libraries generated with Evercode Whole Transcriptome v2 kits require 150 cycle kits for Illumina sequencing. Note that 150 cycle kits actually include reagents for 168 total cycles.

Evercode WT v2 sequencing libraries should be diluted and denatured according to the instruction for the relevant sequencing instrument. We strongly recommended adding 5% PhiX for optimal sequencing quality. 

Depending on the kit configuration, Evercode WT v2 libraries may have been prepared with unique dual indices (UDIs) or with single indices. The tables below outline the cycling requirements for each configuration.

Run configuration requirements for Evercode WT v2 sequencing libraries prepared with UDIs

Libraries should be sequenced with paired reads using the following read structure.

Read Structure of Unique Dual Indexed Evercode Whole Transcriptome v2 Sequencing Libraries

The WT sequence is depicted in gray (between R1 and BC1). A final PCR amplifies the fragmented cDNA and appends the fourth DNA barcodes, UDIs from the UDI Plate - WT, as well as the P5 and P7 adaptors.

Run configuration requirements for Evercode WT v2 sequencing libraries prepared with single indexes

Read Structure of Single Indexed Evercode Whole Transcriptome v2 Sequencing Libraries

The WT sequence is depicted in gray (between R1 and BC1). A final PCR amplifies the fragmented cDNA and appends the fourth DNA barcode as well as the P5 and P7 adaptors.

Additional Resources

For UDI sequences see: Unique Dual Indices (UDI) Sequences

For single index sequences see: Single Index Sequences