High resolution HLA typing using current sequencing-based methods requires time consuming and expensive strategies to distinguish between ambiguous alleles or allele combinations. Next Generation Sequencing (NGS) approaches have been developed within the last few years that provide allele phase and unambiguous pairwise combinations of alleles. These technologies can reduce the cost of high resolution typing to that of commonly used low or medium-resolution methods while maintaining equivalent simplicity in methods and application.
Scisco Genetics has developed a system using the Illumina Miseq system, which is based on a sequencing-by-synthesis approach utilizing fluorescently labeled reversible terminator nucleotides. After assay specific amplification, samples are tagged with unique indexes and pooled together and applied to the MiSeq device where they are amplified as individual clusters and sequenced using universal sequencing primers. The result is several million reads that can be analyzed using Scisco Genetics software to report unambiguous HLA allele types and KIR haplotypes for scores to hundreds of individual samples simultaneously.
Our state-of-the-art technology and automated workflow allows us to provide you with high resolution typing for hundreds of samples in one sequencing run.
First, exons from both HLA and KIR genes are amplified with locus-specific primers. The copy number of KIR genes are monitored with additional amplification reactions. PCR products are then tagged with unique index sequences that allow samples to be pooled and sequenced together.