Challenges in obtaining high-quality data from a custom-made panel for the next generation sequencing (NGS) using Ion Torrent GeneStudio™ S5 platform

Authors

  • Lana Salihefendić Alea Genetic Center, Bosnia and Herzegovina and Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International Burch University, Bosnia and Herzegovina
  • Adna Ašić Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International Burch University, Bosnia and Herzegovina
  • Ivana Čeko Alea Genetic Center, Bosnia and Herzegovina
  • Dino Pećar Alea Genetic Center, Bosnia and Herzegovina
  • Larisa Bešić Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International Burch University, Bosnia and Herzegovina
  • Naida Mulahuseinović Alea Genetic Center, Bosnia and Herzegovina
  • Amra Džuho Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International Burch University, Bosnia and Herzegovina
  • Laura-Severina Köhn Department of Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International Burch University, Bosnia and Herzegovina
  • Rijad Konjhodžić Alea Genetic Center, Bosnia and Herzegovina

Keywords:

COVID-19, next-generation sequencing, Ion Torrent GeneStudio™ S5, immunological response

Abstract

The goal of this part of the study was to optimize the sequencing procedure for 16 human genes and their regulatory regions that might be associated with differential immunological response to COVID-19. The study was performed on 60 COVID-19 patients from the General Hospital of Tešanj, Bosnia and Herzegovina, categorized into three groups of mild, moderate, and severe clinical manifestation, based on the diagnosis by the residential physician. Target coding sequences and their regulatory regions were amplified for the following genes: HLA-A, HLA-B, HLA-C, ACE2, IL-6, IL-4, TMPRSS2, IFITM3, IL-12, RIG-I/DDX58, IRF-7, IRF-9, IL-1B, IL-1A, CD55, and TNF-α. DNA was isolated from the whole blood samples stored at -20°C for six months using QIAamp® DNA Mini Kit according to manufacturer’s instructions. Since NGS analysis of target genomic regions was performed on the Ion Torrent GeneStudio™ S5 platforms, libraries were prepared using Ion AmpliSeq™ Library Kit Plus according to manufacturer’s instructions in a protocol optimized for low-quality DNA. Due to dissatisfactory sequencing results, further protocol optimization steps were employed through separating two primer pools, increasing the number of PCR cycles, and decreasing the annealing temperature for the primer pool which showed poorer amplification results. In the end, 36 samples produced optimal results, while the remaining 24 samples will be re-sequenced following repeated sample collection and DNA isolation, accompanied by additional protocol modifications.

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Published

2022-12-16

Issue

Vol. 2 No. 2 (2022); Journal of Bioanthropology

Section

Research paper

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