No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing

Area of Science:

• Genomics
• Immunogenetics
• Molecular Biology

Background:

• MHC class I genes are crucial for immune response and vary significantly between individuals.
• Accurate characterization of MHC class I alleles is essential for nonhuman primate research, including immunology and transplantation studies.
• Previous methods for MHC class I sequencing have limitations in obtaining full-length transcripts.

Purpose of the Study:

• To validate the Single-Molecule Real-Time (SMRT) sequencing technology with circular consensus sequencing (CCS) for accurate identification of MHC class I transcripts.
• To apply the SMRT-CCS method to discover novel MHC class I transcript sequences in Chinese cynomolgus macaques.
• To establish a straightforward protocol for characterizing unfragmented single-molecule cDNA transcripts for MHC class I allele discovery.

Main Methods:

• Utilized Pacific Biosciences (PacBio) RS II platform for SMRT sequencing.
• Employed circular consensus sequencing (CCS) to enhance sequence accuracy by mitigating SMRT's inherent error rate.
• Sequenced MHC class I complementary DNA (cDNA) from Mauritian-origin and Chinese cynomolgus macaques (Macaca fascicularis).

Main Results:

• Validated that SMRT-CCS accurately identifies MHC class I transcripts in Mauritian cynomolgus macaques, consistent with prior sequencing methods.
• Characterized 60 novel full-length MHC class I transcript sequences from a cohort of Chinese cynomolgus macaques.
• Demonstrated the capability of SMRT-CCS to sequence approximately 1100-bp full-length coding regions of MHC class I cDNA.

Conclusions:

• SMRT-CCS is a reliable and accurate method for characterizing full-length MHC class I transcripts.
• This approach offers a significant advancement for MHC class I allele discovery in nonhuman primates.
• The described protocol provides a streamlined method with potential to revolutionize the field of immunogenetics.