NGS Services

Single Cell Analysis

While single cell sequencing can provide valuable insights into the complexity and heterogeneity of the cell types in the context of a tissue or tumor, there are challenges that exist that one needs to consider to be able to extract meaningful data from a single cell experiment. Key challenges during the workflow starts with preparation of single cell suspensions with good cell viability, efficiently capturing the diverse cell types in a mix via the appropriate cell capture method, and additional complexities with the quality of the data generated in a single cell experiment. Due to the tremendous utility of single cell gene expression in both basic as well as translational research, several approaches are now available for optimal isolating & scaling single cells isolation, preparing sequencing ready libraries that capture the maximal transcript diversity in a given cell, and analysis strategies to generate meaningful information from the data. At MedGenome, we have adopted several of the commercially available platforms for single cell analysis including the 10x Genomics Chromium platform and the SMART-Seq chemistries by Takara. As part of our service workflow we inspect the cell suspensions for viability and assess the quality of the sample prior to proceeding with the library preparation. We follow the manufacturer’s recommended guidelines for our quality control in every step of the process and provide customers with accurate QC reports.

Highlights of our services are:

  • Support with experimental design and selection of appropriate single cell workflow
  • Process single cell samples for a wide range of sample types
  • Process single cell sequencing libraries from live and fixed tissues
  • Provide end to end solution in library preparation, sequencing and bioinformatics analysis
  • Provide advanced bioinformatics analysis and data interpretation interpretation of data with advanced bioinformatics analysis solutions and data curation services

Workflows for sample processing and selection of single cell genomics applications at MedGenome

A) Starting with intact single cell / nuclei suspensions obtained from dissociated tissues, primary cells, cell lines or sorted cells, cell viability checks (trypan blue staining and microscopic examination) and dead cell removal steps are performed via washes. B) The rationale for selecting the platform for single cell isolation/encapsulation depends on the starting number of viable cells available, the recovery rate needed and the cell size and shape of the cell. C) At MedGenome, we have integrated and validated two of the well validated approaches. C.1) If there are greater than 100,000 cells available with good viability, and cells are less than 40uM, the 10X Genomics Chromium is recommended. C.2) If cells are larger than 40uM or have low quantities with a few hundred to a few thousand cells available for sequencing, the plate based SMART-Seq technology is an ideal approach to capture the full-length transcriptome diversity.

10X Single Cell Library Prep, Sequencing, and Analysis Service Offerings

Source Library Prep Options Analysis Offering
  • Fresh Cell Suspension
  • Frozen Cells
  • Fixed Cells
  • Single Cell RNA: 3′ GEX, 5’GEX
  • Single Cell TCR, BCR
  • Single Cell ATAC
  • Single Cell CITE – Total Seq A, B, C

Standard and Advanced Analysis

  • 10X CellRanger
  • Seurat QC
  • tSNE
  • Cell hashing
  • Differential gene expression
  • Cell type assignments
  • Advanced VDJ outputs
  • Multi-model overlay of multiple applications

Single Cell Plate Seq Library Prep, Sequencing, and Analysis Service Offerings

Source Library Prep Options Analysis Offering
  • Isolated Cells in 96 well plate(s)
  • Takara SMART-Seq Single Cell kit

Standard Analysis

  • Raw Data, Alignment
  • FPKM Raw Read Count and Expression Estimate Output
  • Hierarchical Clustering Analysis
  • Clustering, PCA, Heatmap Correlation, Splicing Annotation, Gene Body Coverage

Advanced Analysis

  • Differential Expression Outputs – Heat map and Volcano Plots
  • Pathway Analysis, Gene Ontology Output and Figures
  • Isolated T Cells in 96 well plate(s)
  • Takara SMARTer TCR alpha/beta Profiling Kit

Standard Analysis

  • MiXCR Output file – Clonotype Frequency, Gene and Amino Acid Sequences and paired sequences
  • V J Gene Usage, Shannon’s Diversity, V J Circos Plot, Spectratype Plot, Scatter Plot
  • Custom figures available upon request

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