Immune repertoire sequencing: RepSeq and RepSeq+
iRepertoire’s sequencing services RepSeq and RepSeq+ complement our amplification technologies arm-PCR and dam-PCR, respectively. Because arm-PCR and dam-PCR have different strengths, the decision of whether to use RepSeq or RepSeq+ will depend on the nature of your specific project.
Arm-PCR uses internal and external nested primers for extremely inclusive repertoire sequencing; therefore, RepSeq is preferable for the discovery of low-frequency clonotypes, especially when cell counts are low. The arm-PCR workflow is less complex, so arm-PCR can be performed in-house with standard laboratory equipment, whereas dam-PCR must be completed by iRepertoire. For more information about arm-PCR products, please see our products page.
RepSeq+ provides a more quantitative picture of the entire immune repertoire with unprecedented multiplexing abilities. RepSeq+ is the only platform that allows for simultaneous amplification and sequencing of all 7 TCR and BCR chains. Additionally, because unique molecular identifiers can be used in dam-PCR, RepSeq+ provides for more sensitive detection and correction of PCR and sequencing errors. When it is important to identify single nucleotide variability with high fidelity, as in B cell receptor hypermutation analysis, RepSeq+ is a better choice. For processing RepSeq+ samples in-house, see our fully automated iR-Flex system.
For more information about arm-PCR and dam-PCR, see our amplification technologies post.
iRepertoire’s amplification technologies and sequencing services are designed to be compatible with multiplexing so you can save costs by batch processing multiple samples into one sequencing run. If your order is not large enough to fill an entire flow cell, we can use other customer samples to fill the remaining space.
For example, let’s say you wanted to study the TCR alpha and TCR beta repertoire of a single sample using our RepSeq service. This is equivalent to two barcoded libraries because two unique reagent systems are required for the amplification (one per chain). Sequencing would most likely be performed on the Illumina MiSeq with a V2-500 cycle kit, and because this kit can sequence up to 10 samples at a read depth of 1 million reads per sample, we would need to place eight other customer samples in your sequencing run to complete the flow cell.
Fewer samples (i.e., not filling an entire flow cell) could mean an extended time frame for sequencing to complete since the remaining space needs to be filled. Alternatively, if you have enough libraries to fill an entire sequencing lane, then this delay will not occur. If your libraries are multiplexed with other customer’s libraries, we can provide the de-multiplexed data (not the raw sequencing data). This restriction does not apply if you fill an entire lane with only your samples.
For more information about multiplexing, see our page on pooling samples.
For more information about iRepertoire’s reagents, see the learning center articles about primer systems.