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お問い合わせ

真のHLAアリル多様性を解明

HLA遺伝子における、タンパク質結合領域以外の多型の特徴づけは関連解析に重要です1。しかしこの領域は配列の相同性が高く、また多型が高頻度に発生するため、真のアリル多様性の決定はこれまで非常に困難な作業でした。シークエンスがHLAタイピングに広く使われるツールとなった今でも、二倍体の曖昧さ、およびインピュテーションへの依存は現在も課題となっています。特にアリル間の配列相同性領域が長ければ長くなるほど、既存の手法では解析が困難です2

HLA遺伝子にわたり包括的に多型にアクセス

PacBioの1分子リアルタイム(SMRT) シークエンスを使えば、免疫に関与する疾患の原因探索、造血移植における移植片対宿主病、そして薬剤高感受性への理解を深めることができます。

SMRTシークエンスは1分子単位で観察する10-15 Kbのリードで構成されており、HLAクラスIおよびII遺伝子の大部分をシークエンスすることができます。この技術は完全長HLA遺伝子にわたる高精度なコンセンサスシークエンス(QV50実証)を産出でき、インピュテーションをすることなしに、リファレンスを必要とせず直接フェーズ化および高解像度のHLAタイプを行うことができます2

このアプリケーションの特長は下記を含みます。

  • コスト効率の良いマルチプレックスソリューションでフレキシブルにプロジェクトサイズを拡張(例:エクソン2, 3, 4、あるいはHLA遺伝子全体をフェーズ化)
  • HLA遺伝子のアリル特異的なSNPが存在しない領域においてもロングリードでフェーズ化
  • インピュテーションなしにアリルレベルで区別
  • 5′ UTR、イントロン、3′ UTRなど遺伝子制御領域における多型を検出
  • がんや転写産物など、ポリクローナル化されたサンプルにおいても低頻度変異の特長づけ
  • リファレンスフリーのde novoコンセンサスシークエンス産出で新しいHLAアリルの検出

ワークフロー:ターゲット領域から高精度な多型とハプロタイプ

 

 

ライブラリー調製

 

 

PacBioシステムでSMRTシークエンス

 

SMRT AnalysisPacBio DevNetでデータ解析

 

特集:HLAクラスII遺伝子のための完全な遺伝子リファレンスを構築

Immunologists at DKMSの免疫学者たちは、HLA-DPB1の完全遺伝子におけるリファレンスフリーのコンセンサス配列を決定しました。この研究では、IGMT / HLAデータベースには、550の既知HLA-DPB1アリルから構成された12の完全長リファレンスがありました。わずか1回のSMRTシークエンスランで、研究sh田たちは9つの新しいアリルを発見し、さらに48検体の完全にフェーズ化されたクラスII HLAジェノタイプを実施しました3論文を読む

SMRTシークエンスがどのようにインピュテーションなしのHLAタイピングを可能にするか、詳細はお問い合わせください。

References

  1. Hosomichi, K., et al., (2015) The impact of next-generation sequencing technologies on HLA research. Journal of Human Genetics. 60 (11), 665–673.
  1. Nelson, W. C., et al., (2015) An integrated genotyping approach for HLA and other complex genetic systems. Human Immunology. 76 (12), (928-938).
  1. Lang, K. et al. (September, 2015) Phased full-length SMRT Sequencing of HLA DPB1. Poster presented at 41st Annual Meeting of the American Society for Histocompatibility and Immunogenetics. Savannah, GA..

Selected Resources