Seamless Integration and Impact of the G4™ Sequencing Platform: A Case Study Using Parse Biosciences Evercode™ WT Libraries for Single Cell RNA Sequencing

July 31, 2023

Single cell RNA sequencing (scRNA-Seq) holds great potential for revolutionizing research and clinical studies. Nevertheless, conducting complementary high-throughput sequencing for these endeavors entails substantial upfront costs and the need for costly materials. The G4™ Sequencing Platform offers a solution that empowers researchers to obtain affordable and precise scRNA-seq results, with more speed and flexibility than other sequencers on the market.  The G4™ Sequencing Platform was designed with operational flexibility and efficiency in mind, seamlessly integrating into existing next-generation sequencing (NGS) workflows. This plug-and-play capability is enabled by partnerships with industry leading library preparation partners and data analysis providers. 

Teaming Up with Parse Biosciences for Highly Flexible and Scalable scRNA-Seq

The Parse Biosciences Evercode™ Whole Transcriptome (WT) kits provide a solution for scalable single cell RNA sequencing that utilizes the Evercodea split pool combinatorial barcoding approach for library preparation. Evercode WT products accommodate studies of any size with kits that process up to 10,000 cells (WT Mini), 100,000 cells (WT), and 1 million cells (WT Mega). With the ability to fix and store cells or nuclei at different times, users can run experiments in parallel for added flexibility. The kit does not require the use of microfluidics equipment and generates data of high specificity. The Evercode WT kit includes access to data analysis software and Parse Biosciences’ scientist support.

Libraries prepared with the Parse Biosciences Evercode WT kit can be easily adapted for sequencing on the G4 Platform using Singular Genomics PCR primers; combined with G4’s flexible run sizes, laboratories can maximize operational efficiency and reduce sample turnaround times.

Sequencing Evercode Libraries on the G4

Evercode WT scRNA-Seq libraries were prepared from frozen peripheral blood mononuclear cells (PBMCs) collected from four healthy donors. A portion of the library was converted for sequencing on the G4 using the G4 Non-Indexed Library Prep Primers, while a second portion of the library was sequenced on the Illumina NovaSeq® 6000. Resulting FASTQ files from each sequencer were processed using the Parse Biosciences data analysis and bioinformatics pipeline.

Data analysis revealed a high concordance of results between the sequencing platforms with respect to read quality and estimated cell counts. Both uniform manifold approximation and projection (UMAP) and Leiden clustering demonstrated nearly identical grouping of single cell transcriptomes, strongly correlated bulk transcription profiles, and matched T-Cell, B-Cell, and Monocyte gene expression profiles between the G4 and NovaSeq 6000 systems (Figure 1). 

Dr. Charlie Roco, Ph.D, Co-Founder and Chief Technology Officer for Parse Bioscience, further echoed the powerful combination these performance data,  “With a rapid turnaround, the G4 platform recovered high-quality data that our computational software was able to read and use to produce immediate insights.”

Figure 1.(A) Unsupervised Leiden clustering for the G4 and NovaSeq 6000 datasets overlaid on the UMAP embedding. (B) Heatmap of average gene expression profiles of a panel of PBMC phenotyping markers for each cell type.

A Winning Combination

The two different read densities of the F2 and F3 flow cells offer users the option to select a flow cell that matches sample throughput, eliminating the requirement of pooling samples to fill a large flow cell. By reducing reagent waste and unused flow cell capacity, sequencing becomes more cost-effective.