Targeted Next Generation Sequencing Panel Design in Complex Monogenic Diseases: The Example of Primary Immunodeficiency

Abstract

Objective: Next-generation sequencing technologies can generate an analysis of a large number of candidate genes, all the coding regions of a genome, or whole genomes with a high degree of accuracy and within a short amount of time. Targeted next generation sequencing systems have been found in many routine genetic diagnosis applications that allow the sequencing of only the candidate regions of the genome. Materials and Methods: In this study, we designed PCR-based targeted next generation sequencing (NGS) panels for severe combined immunodeficiency (SCID) and primary antibody deficiency (PAD) and created an algorithm for analysing high-throughput data. Results: We screened 112 patients (48 SCID and 64 PAD) and we detected genetic variations in 58% of the SCID and in 14.2% of the PAD patients. All variants were validated by Sanger sequencing to validate the accuracy of the NGS panel and analysis algorithm. Conclusion: Designing targeted next generation sequencing panels with an appropriate method, in accordance with the targeted region, and analysing the raw data with a suitable workflow, increases the success of the panel.

Keywords

• Next generation sequencing
• variant annotation
• targeted-ngs panel design

Tanısı Zor Tek Gen Hastalıklarında Hedefe Yönelik Yeni Nesil Dizileme Panel Tasarımı: Primer İmmün Yetersizlik Örneği

Abstract

Amaç: Yeni nesil dizileme teknolojileri bugün çok sayıda aday genin, genomun tüm kodlayan bölgelerinin hatta tüm genomun analizini tek seferde ve kısa süre içerisinde düşük maliyet ve yüksek hassasiyette, güvenilir bir şekilde mümkün kılmaktadır. Hedefe yönelik yeni nesil dizileme sistemleri genomda sadece belirli bölgenin dizilenmesine imkan veren, tüm genom dizilemelere göre uygulaması ve analizi daha kolay, hızlı ve yüksek güvenirlilikte bir yöntem olarak pek çok rutin genetik tanı uygulamalarında yerini bulmuştur. Gereç ve Yöntem: Çalışmamızda primer immün yetersizliklerin en yaygın grubu olan primer antikor yetersizlikleri (PAY) ve en ağır seyirli grubu ağır kombine immün yetersizlikler (AKİY) için hastalık ile ilişkili olduğu bilinen genleri kapsayan PZR temelli genetik tanı panelleri geliştirilmiş ve ortaya çıkan yüksek verinin yorumlanması için bir analiz akışı oluşturulmuştur. Bulgular: Tasarlanan paneller ile toplam 112 hasta (PAY:64, AKİY:48) dizilenmiş ve AKİY hastalarının %58’i ve PAY hastalarının %14,2’sinde hastalık ile ilişkili varyantlar tespit edilmiştir. Tüm varyantlar Sanger dizileme ile doğrulanarak oluşturulan moleküler tanı panellerinin ve analiz algoritmasının doğruluğu kontrol edilmiştir. Sonuç: Hedefe yönelik yeni nesil dizileme panellerinin hedeflenen bölgeye uygun olarak doğru yöntemle tasarlanması ve çıkan ham datanın doğru iş akışı ile analiz edilmesi panelin başarısını arttırmaktadır.

Keywords

• yeni nesil dizileme
• varyant yorumlama
• hedefe yönelik panel tasarımı

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