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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.

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Related Experiment Video

Updated: May 14, 2026

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
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High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon

P V Moonsamy1, T Williams, P Bonella

  • 1Roche Molecular Systems, Inc., Pleasanton, CA, USA. Priscilla.Moonsamy@roche.com

Tissue Antigens
|February 13, 2013
PubMed
Summary

High-throughput human leukocyte antigen (HLA) genotyping is achieved using next-generation sequencing and multiplex identifiers. This method enables accurate HLA typing for bone marrow donor registries, improving donor selection efficiency.

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Area of Science:

  • Immunogenetics
  • Genomics
  • Molecular Biology

Background:

  • Human leukocyte antigen (HLA) loci are highly polymorphic, making accurate genotyping challenging.
  • Efficient HLA typing is crucial for unrelated bone marrow donor registries to ensure successful transplantation.
  • Existing methods often require high sample throughput and low cost per sample.

Purpose of the Study:

  • To develop and validate a high-resolution, high-throughput HLA genotyping method.
  • To leverage next-generation sequencing and multiplex identifiers for accurate HLA allele identification.
  • To assess the feasibility of this method for donor registry applications.

Main Methods:

  • Utilized the 454 Genome Sequence (GS) FLX System with Conexio Assign ATF 454 software for sequencing.
  • Employed the Fluidigm Access Array System for multiplexing 48 samples with unique multiplex identifiers (MIDs).
  • Generated 2304 parallel PCRs using a 4-primer system incorporating MIDs and 454 adaptor sequences for amplicon generation.

Main Results:

  • Achieved 100% concordance with known genotypes for 192 samples using 8 primer pairs and 96 MIDs in a single GS FLX run.
  • Obtained an average of 166 sequence reads per amplicon for initial genotyping.
  • Successfully performed high-resolution genotyping on 96 samples using 14 primer pairs, yielding an average of 173 reads per amplicon.

Conclusions:

  • The described method provides high-resolution, high-throughput HLA genotyping.
  • This approach is accurate and efficient for typing multiple HLA loci.
  • The technology is suitable for applications in bone marrow donor registries, enhancing donor selection processes.