- de novo Sequencing
- Whole Genome Resequencing
- Exome Sequencing
- Target Region Sequencing
- Genotyping by Sequencing
- Whole Genome Mapping
- Sanger Sequencing
- Single-Cell DNA Sequencing
- Human MHC-Seq
- Single-Cell Sequencing
- Immune Repertoire Sequencing
- FFPE Samples
Immune repertoire refers to the sum total of functionally diverse B and T-cells in the circulatory system at any given moment (Wang et al. 2011). The diversity of immune repertoire is of vital importance for health. Immune Repertoire Sequencing (IR-SEQ) is to amplify the complimentary determining region (CDR) of B-cell receptor (BCR) or T-cell receptor (TCR) using multiple-PCR or 5’ RACE methods, followed by high-throughput sequencing. It is used to study the diversity of the immune system and the associations between immune repertoire and diseases.
- Proprietary methods: we have designed specific primers for CDR3 V region and C region to amplify CDR3 region, and patents for TRA, IGH, and IGK/IGL have been applied successfully.
- High-fidelity amplification methods: multiple-PCR or 5’ RACE method is used to equivalently amplify different clones and the number of unique clones is as large as 105.
- Multiple sequencing platforms: Hiseq2000, Hiseq2500 and Roche 454 are available for different project requirements.
- Vaccine development and efficacy
- Biomarker discovery
- Minimal residual disease detection
- Investigation of autoimmune diseases
- Transplant rejection and tolerance
1. C. Wang et al. J. Immunol. 186, 65.20 (2011)
Workflow:Peripheral blood mononucleated cells (PBMCs) are isolated from peripheral blood, followed by DNA or RNA extraction. Multiple-PCR or 5’RACE method is used to capture CDR3 region (5’ RACE can also capture CDR1 and CDR2), and then the captured region is sequenced on high-throughput platforms (Hiseq2000, Hiseq2500, Miseq or Roche 454).
Basic data processing
- Data filtering, removal of adapter contamination and low quality reads from raw reads
- Merge paired reads and remove background noise
- Output data statistics and evaluation of contents and quality of sequencing data
- Alignment to V/D/J gene segments separately in IMGT database
- Realignment for best result
- CDR sequence and base composition
- V/D/J recombination insertion and deletion
- Translate CDR sequences into peptides
Immune repertoire profiling
- IR expression profiling
- Diversity visualization, 2D and 3D histogram of V-J pairing
Expression differential analysis
- Differential analysis of diversity between samples (e.g. Simpson index, Shannon-Weaver index)
- Differential expression analysis of clones between samples (e.g. CDR3, V-D-J)
- Differential expression analysis of clones between groups (e.g. CDR3, V-D-J)
Customized bioinformatics analysisWe can perform other customized analyses to meet specific requirements.
- Purity: OD260/280=1.8~2.0, without degradation and RNA contamination
- Concentration: c ≥ 37.5ng/μL
- Quantity: 2.5 μg ≤ amout < 5 μg (for library construction once); amount ≥ 5 μg (for library construction more than twice)
- Purity: RIN ≥ 7.0, 28S/18S ≥ 1.0, the baseline is smooth and 5S peak is normal
- Concentration: c ≥ 200ng/μL
- Quantity: 5 μg ≤ amount