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.

Cancer is an abnormal proliferation of cells that is characterized by the presence of genomic alterations including DNA mutations, deletions, insertions, translocations, inversions, and others. KRAS gene is one of the most mutated genes across different cancer types. The most common mutations in KRAS are found in codons 12 and 13 of KRAS protein, which are associated with a lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapy. This study assessed and compared the performance between two diagnostic methods: droplet digital PCR (ddPCR) and next generation sequencing (NGS). The main goal was to determine KRAS G12 / G13 mutant allele fraction using NGS and to compare the accuracy toddPCR. A total of 28 samples of non – small cell lung cancer (NSCLC) and colorectal cancer (CRC) were analyzed using ddPCR and NGS methods. Our results show that even though both methods exhibited high rate of concordance and correlation, the study proved that ddPCR is more superior when it comes to detecting low frequency mutations. Even though strong correlation was observed, based on the values obtained, we concluded that ddPCR is more accurate, reliable, and sensitive in comparison with NGS.

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

Bosnia and Herzegovina is located in the South-Eastern Europe, characterized by numerous historical influences, massive migration processes and complex population structure. For that reason, the aim of this study is to provide an accurate and precise update of the population genetics data of allele frequencies on 23 Y-STR loci in Bosnia and Herzegovina using larger sample size. For this purpose, 480 adult male individuals from the general population have been genotyped over 23 Y-STR loci contained in the PowerPlex Y23 system. Population genetics parameters have been calculated, namely allele and haplotype frequencies, gene and haplotype diversity, as well as Rst and P values for the assessment of interpopulation differences. The obtained results are in close agreement with previously published data for Bosnian-Herzegovinian population, as well as for local subpopulations. This study offers significantly increased resolution and information content, with 454 unique haplotypes. Population comparison reveals no statistically significant differences between the study population and 12 European populations used for comparison, as visualized through an MDS plot and neighbour-joining phylogenetic tree. This study offers representative data for local Y chromosomes that can be used for forensic applications, paternity and kinship testing, as well as for genealogical studies.

Human Y-chromosomal haplogroups are an important tool used in population genetics and forensic genetics. A conventional method used for Y haplogroup assignment is based on a set of Y-single nucleotide polymorphism (SNP) markers deployed, which exploits the low mutation rate nature of these markers. Y chromosome haplogroups can be successfully predicted from Y-short tandem repeat (STR) markers using different software packages, and this method gained much attention recently due to its labor-, time-, and cost-effectiveness. The present study was based on the analysis of a total of 480 adult male buccal swab samples collected from different regions of Bosnia and Herzegovina. Y haplogroup prediction was performed using Whit Athey’s Haplogroup Predictor, based on haplotype data on 23 Y-STR markers contained within the PowerPlex® Y23 kit. The results revealed the existence of 14 different haplogroups, with I2a, R1a, and E1b1b being the most prevalent with frequencies of 43.13, 14.79, and 14.58%, respectively. Compared to the previously published studies on Bosnian-Herzegovinian population based on Y-SNP and Y-STR data, this study represents an upgrade of molecular genetic data with a significantly larger number of samples, thus offering more accurate results and higher probability of detecting rare haplogroups.

Introduction Serological detection of SARS-CoV-2-specific immunoglobulins G (IgG) and M (IgM) antibodies is becoming increasingly important in the management of the COVID-19 pandemic. Methods We report the first results of COVID-19 serological testing in Bosnia and Herzegovina on 2841 samples collected and analysed in 2 medical institutions in Sarajevo. Antibody detection was performed using commercially available kits. Results In the first cohort, 43 IgM-positive/IgG-negative and 16 IgM-positive/IgG-positive individuals were detected, corresponding to 3.41% of participants having developed antibodies. In the second cohort, 4.28% participants were found to be IgM-negative/IgG-positive. Conclusions Our results suggest the need for population-wide serological surveying in Bosnia and Herzegovina.

Whole Genome Sequence of four samples from COVID-19 outbreaks was done in two laboratories in Bosnia and Herzegovina (Veterinary Faculty Sarajevo and Alea Genetic Center). All four BiH sequences cluster mainly with European ones (Italy, Austria, France, Sweden, Cyprus, England). The constructed phylogenetic tree indicates probable multiple independent introduction events. The success of future containment measures concernig new introductions will be highly challenging for country due to the significant proportion of BH population living abroad.

Next Generation Sequencing (NGS) has become powerful tool in molecular oncology. It allows multiparallel targeted sequencing that enables comprehensive assessment of tumor heterogeneity. Detection of mutations in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) defines patients diagnosis, therapy and prognosis. Multiple genes, their somatic mutations to be precise, carry different degrees of importance for any of these stages. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which was used in this study, allows detection of hotspot mutations on 22 genes in a single reaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequency mutation detection and it has been used as a validation method for mutations that were detected with NGS. It has high sensitivity and enables accurate detection of mutant allele in a background of abundant wild type alleles. For this study 35 samples of CRC and NSCLC were sequenced and same samples were analysed on ddPCR for KRAS, NRAS, EGFR and BRAF genes. All processed samples were successfully sequenced and had average base coverage >500X. NGS sequencing proved itself to be cost effective, has shorter turnaround time and is highly sensitive. Out of 35 samples, 25 had genetic alterations, while 10 samples are reported as wild type but were still tested on ddPCR as controls. In three samples low frequency somatic mutations were detected by NGS and verified using ddPCR, which leads us to conclusion that ddPCR is a good tool for verification of somatic mutations in CRC and NSCLC.