Sequencing Depth
In general, targeting 20,000 reads per cell is a good place to start for whole transcriptome (WT) sequencing depth optimization. However, the ideal sequencing depth will be dependent on your sample type and experimental goals. For example, if you are aiming to sequence rare cell types in a heterogenous population, you will likely need to sequence much deeper than this. Alternatively, if you are analyzing a homogenous population, you may not need to sequence as deeply.
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 allocate different numbers of cells or nuclei into each sublibrary. We recommend creating one small sublibrary (e.g., 500 or 1,000 cells) that can be sequenced 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
Parse WT libraries require a 150 cycle kit minimum (which will get you up to 168 total cycles as Illumina includes extra reagents as a standard practice). WT 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.
Run configuration requirements for WT sequencing libraries prepared with UDIs
Libraries should be sequenced with paired reads using the following read structure.
| Read | Cycles |
| Read 1 | 66 |
| i7 Index (Index 1) | 8 |
| i5 Index (Index 2) | 8 |
| Read 2 | 86 |
WT Sequencing Amplicon with UDIs:
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 WT sequencing libraries prepared with single indexes
Libraries should be sequenced with paired reads using the following read structure.
| Read | Cycles |
| Read 1 | 74 |
| i7 Index (Index 1) | 6 |
| i5 Index (Index 2) | 0 |
| Read 2 | 86 |
WT Sequencing Amplicon with Single Index:
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