Pengembangan Metode In-House HLA-Typing Gen HLA Kelas I (HLA A, HLA B, dan HLA C) Menggunakan Next Generation Sequencing Illumina MiSeq

Rika Yuliwulandari, Kinasih Prayuni, Kenconoviyati Kenconoviyati, R. W. Susilowati, Abdul Salam M. Sofro

Abstract


Human leucocyte antigen (HLA) adalah protein penyaji antigen yang lokus genetiknya berada di kromosom 6p21 dengan ukuran sebesar 3,8 Mb dan berasosiasi dengan lebih dari 100 penyakit berbeda yang  kebanyakan merupakan penyakit autoimun. Proses HLA-typing menggunakan sekuensing Sanger masih memberikan ambiguitas terhadap determinasi alel, low-throughput, dan membutuhkan biaya besar untuk sampel dalam jumlah besar. Next generation sequencing (NGS) menjadi metode yang dapat mengatasi kelemahan sekuensing Sanger. MiSeq dari Illumina merupakan salah satu NGS yang digunakan untuk HLA-typing. MiSeq memberikan kemudahan preparasi dan fleksibilitas metode yang dapat dikembangkan sesuai dengan kebutuhan laboratorium penelitian. Penelitian dilakukan di Laboratorium Molekular Genetik, Laboratorium Terpadu Universitas YARSI pada empat orang mahasiswa Fakultas Kedokteran Universitas YARSI etnik Melayu selama periode Mei–Desember 2014. Hasil menunjukkan terdapat total 546 SNP heterozygous, 888 SNP homozygous, 25 insersi, dan 23 delesi dari keseluruhan 11 sampel amplikon dengan coverage 2.106,536x dengan 2x25 siklus pembacaan. Optimasi metode HLA-typing dapat dikatakan berhasil dengan mengombinasikan long-range PCR dan pemilihan ukuran library 300–600 bp. [MKB. 2015;47(3):152–159]

Kata kunci: HLA Kelas I, MiSeq, next generation sequencing


Development of Class I HLA Gene In-House HLA-Typing Methods (HLA A, HLA B, and HLA C) using Next Generation Sequencing Illumina MiSeq


Abstract

Human leukocyte antigen (HLA) is a 3.8 Mb protein presenting antigen whose genetic locus is located in chromosome 6p21 area and have association with more than 100 different diseases that are mostly autoimmune diseases. HLA-typing process using Sanger sequencing still  creates ambiguity in the  determination of  alleles, low-throughput, and costly as it requires a large quantity of sample. Next generation sequencing (NGS) is a method that can overcome the drawbacks of Sanger sequencing. MiSeq Illumina is one of the NGSs that are used for HLA-typing. This study was conducted at the Laboratory of Molecular, Universitas YARSI in a period from May to December 2014. MiSeq provides convenience and flexibility in the preparation methods that can be developed according to the needs of the research laboratory. The results showed that there were a total of 546 SNPs that were heterozygous, 888homozygous SNP, 25 insertions and 23 deletions from the overall 11 amplicon samples with an average coverage with 2x25 read length of  2,106,536x. Our protocol generates good result as we combined long PCR amplicon and size selection method to 300–600 bp fragment.  [MKB. 2015;47(3):152–159]

Key words: HLA Class I, MiSeq, next generation sequencing

 

DOI10.15395/mkb.v47n3.389


Keywords


Medicine

Full Text:

PDF

Refbacks

  • There are currently no refbacks.





 

This Journal indexed by

             


Creative Commons License
MKB is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License

 


View My Stats