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Related Concept Videos

Next-generation Sequencing03:00

Next-generation Sequencing

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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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Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing
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Development and validation of a sample sparing strategy for HLA typing utilizing next generation sequencing.

Denise M McKinney1, Zheng Fu2, Lucas Le1

  • 1Department of Vaccine Development, La Jolla Institute for Allergy and Immunology, 9820 Athena Circle, La Jolla, CA 92037, USA.

Human Immunology
|June 2, 2015
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Summary

This study presents a new method for human leukocyte antigen (HLA) typing using whole genome amplification, enabling accurate results from minimal cell samples. The HLATyphon pipeline significantly increases throughput for HLA genotyping.

Keywords:
HLA typingNext generation sequencingPipelineSample sparing

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Area of Science:

  • Immunogenetics
  • Genomic Medicine
  • High-throughput Sequencing

Background:

  • Accurate human leukocyte antigen (HLA) genotyping is crucial for transplantation and disease association studies.
  • Limited cellular material often restricts the feasibility of traditional HLA typing methods.
  • Existing methods can be costly and time-consuming, limiting large-scale applications.

Purpose of the Study:

  • To develop a general, sample-sparing methodology for comprehensive HLA class I and class II genotyping.
  • To validate the method's accuracy and efficiency for both low-input samples and high-throughput applications.
  • To provide an accessible computational pipeline (HLATyphon) for HLA data analysis.

Main Methods:

  • Whole Genome Amplification (WGA) was employed to enable HLA typing from as few as 300 cells.
  • Next-generation sequencing (NGS) was utilized for HLA-A, -B, and -C genotyping, validated against International Histocompatibility Working Group (IHWG) cell lines.
  • Novel primer strategies targeting intronic regions were developed for HLA-DQ, -DP, and -DRB1 loci, alongside multiplexing strategies for increased throughput.

Main Results:

  • High concordance rates of 99% were achieved for both HLA class I and class II loci compared to established typing data.
  • The methodology demonstrated utility with minimal cell input (300 cells), ideal for sample-limited studies.
  • Multiplexing 96 samples per run increased throughput approximately 8-fold, reducing costs and enhancing efficiency.

Conclusions:

  • A robust and versatile methodology for HLA class I and II genotyping has been established, suitable for low-input samples.
  • The HLATyphon pipeline offers a validated, high-throughput solution for HLA typing, improving accessibility and efficiency.
  • This approach significantly advances the potential for large-scale genetic studies involving HLA polymorphisms.