Optimizing template preparation protocol for targeted massively parallel sequencing (MPS) of PKD1 and PKD2 genes: Illustrative data

Authors

  • Ratchadaporn Chanayat Graduate Program in lmmunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Ekkapong Roothumnong Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Duangkamon Bunditworapom Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Chanin Limwongse Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Kriengsak Vareesangthip Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Manop Pithukpakorn Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
  • Wanna Thongnoppakhun

DOI:

https://doi.org/10.14456/gag.2019.4

Keywords:

ADPKD; PKD1; PKD2; massively parallel sequencing (MPS); long-range PCR (LR-PCR)

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited nephropathy mainly resulted from mutations in PKD1 and PKD2 genes. Mutation analysis of PKD1 gene is challenging because of the huge size and complicated structure which has six pseudogenes with a 97.7% identical with its exons 1-33. A traditional molecular analysis of the two genes via long-range PCR (LR-PCR) amplification followed by Sanger sequencing is still laborious and expensive.  The introduction of massively parallel sequencing (MPS) allows rapid genotyping.  As a prerequisite for successful MPS, sample preparation should have high-quality template libraries and sufficient yields.  This study aimed to optimize the template preparation protocol for successful targeted MPS of PKD1 and PKD2 genes, in order to increase efficiency of ADPKD molecular diagnosis.  The enriched templates, 24 LR-PCR amplicons from both genes (between 2,278 and 8,040 bp) obtained from patient samples carrying either PKD1 or PKD2 mutations, were applied for library preparation using QIAseq FX DNA library kit for which some particular optimizations were needed. Enzymatic fragmentation times were adjusted to be longer than that recommended by the manufacturer.  In addition, amplification-free library preparation was chosen to minimize the chance of allele-dropout phenomenon usually occurring in PCR reaction especially in case of high-GC amplicons, thus the library amplification was omitted before being subjected to sequencing by the Miseq Illumina platform. Two representative data for both genes showed corresponding mutations to those identified by the previous Sanger sequencing with high coverage read depths that reflected high-quality libraries and sufficient yields.  These preliminary results demonstrated that the optimized library preparation protocol for targeted MPS of PKD1 and PKD2 based on the templates from high-GC LR-PCR amplicons is able to detect the specified variants identified by Sanger sequencing with 100% concordance, being an effective method for simultaneous mutation analysis of both ADPKD-causing genes.

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Published

2019-08-26

Issue

Vol. 12 No. 1 (2019): JAN - APR 2019

Section

Research Articles