Genetic Characterization of Molecular Targets in Korean Patients with Gastrointestinal Stromal Tumors

PURPOSE: Gastrointestinal stromal tumors (GISTs) frequently harbor activating gene mutations in either KIT or platelet-derived growth factor receptor A (PDGFRA) and are highly responsive to several selective tyrosine kinase inhibitors. In this study, a targeted next-generation sequencing (NGS) assay with an Oncomine Focus Assay (OFA) panel was used for the genetic characterization of molecular targets in 30 Korean patients with GIST.MATERIALS AND METHODS: Using the OFA that enables rapid and simultaneous detection of hotspots, single nucleotide variants (SNVs), insertion and deletions (Indels), copy number variants (CNVs), and gene fusions across 52 genes relevant to solid tumors, targeted NGS was performed using genomic DNA extracted from formalin-fixed and paraffin-embedded samples of 30 GISTs.RESULTS: Forty-three hotspot/other likely pathogenic variants (33 SNVs, 8 Indels, and 2 amplifications) in 16 genes were identified in 26 of the 30 GISTs. KIT variants were most frequent (44%, 19/43), followed by 6 variants in PIK3CA, 3 in PDGFRA, 2 each in JAK1 and EGFR, and 1 each in AKT1, ALK, CCND1, CTNNB1, FGFR3, FGFR4, GNA11, GNAQ, JAK3, MET, and SMO. Based on the mutation types, majority of the variants carried missense mutations (60%, 26/43), followed by 8 frameshifts, 6 nonsense, 1 stop-loss, and 2 amplifications.CONCLUSIONS: Our study confirmed the advantage of using targeted NGS with a cancer gene panel to efficiently identify mutations associated with GISTs. These findings may provide a molecular genetic basis for developing new drugs targeting these gene mutations for GIST therapy.

Analysis of CSF1PO and D18S51 Loci Based on Ion Torrent PGM™ Platform / 法医学杂志

OBJECTIVES@#To analyse and detect CSF1PO and D18S51 loci by next generation sequencing （NGS） technology for the study on their sequence polymorphism.@*METHODS@#The peripheral blood samples were collected from 165 unrelated individuals of Chinese Han population. DNA samples were obtained by QIAamp DNA Mini kit. The library was constructed by Ion Plus Fragment Library. DNA sequencing analysis was performed on Ion Torrent PGM™ Platform. The newfound alleles were verified by Sanger sequencing. Data were analysed by Torrent Suite™ v5.0.2 and Integrative Genomics Viewer for the genotype identification and frequency count. The data were analysed statistically by PowerState v12.@*RESULTS@#The length and sequence polymorphisms of CSF1PO and D18S51 loci were simultaneously obtained by NGS technology. A new genotype was found on CSF1PO locus, and two new genotypes on D18S51 locus. Sanger sequencing was used to verify the newfound alleles found by NGS technology, and the results of verification showed consistency.@*CONCLUSIONS@#The structure of core repeats on CSF1PO and D18S51 loci was detected by NGS in this study for the improvement of the identifying performance of locus.

Draft genome sequence of alcohol-tolerant bacteria Pediococcus acidilactici strain K3

Abstract Pediococcus acidilactici strain K3 is an alcohol-tolerant lactic acid bacterium isolated from nuruk, which is a traditional Korean fermentation starter for makgeolli brewing. Draft genome of this strain was approximately 1,991,399 bp (G+C content, 42.1%) with 1525 protein-coding sequences (CDS), of which 44% were assigned to recognized functional genes. This draft genome sequence data of the strain K3 will provide insights into the genetic basis of its alcohol-tolerance.

Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGMTM Platform / 法医学杂志

Objective To analyze and detect the whole genome sequence of human mitochondrial DNA (mtDNA) by Ion Torrent PGMTM platform and to study the differences of mtDNA sequence in different tissues.Methods Samples were collected from 6 unrelated individuals by forensic postmortem examination,including chest blood,hair,costicartilage,nail,skeletal muscle and oral epithelium.Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer.Libraries were constructed with Ion ShearTM Plus Reagents kit and Ion Plus Fragment Library kit.Whole genome sequencing of mtDNA was performed using Ion Torrent PGMTM platform.Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HV Ⅰ region.Results The whole genome sequence of mtDNA from all samples were amplified successfully.Six unrelated individuals belonged to 6 different haplotypes.Different tissues in one individual had heteroplasmy difference.The heteroplasmy positions and the mutation positions on HV I region were verified by Sanger sequencing.After a consistency check by the Kappa method,it was found that the results of mtDNA sequence had a high consistency in different tissues.Conclusion The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues,which have good consistency.The results provide guidance for the further applications of mtDNA in forensic science.

Entire Mitochondrial DNA Sequencing on Massively Parallel Sequencing for the Korean Population

Mitochondrial DNA (mtDNA) genome analysis has been a potent tool in forensic practice as well as in the understanding of human phylogeny in the maternal lineage. The traditional mtDNA analysis is focused on the control region, but the introduction of massive parallel sequencing (MPS) has made the typing of the entire mtDNA genome (mtGenome) more accessible for routine analysis. The complete mtDNA information can provide large amounts of novel genetic data for diverse populations as well as improved discrimination power for identification. The genetic diversity of the mtDNA sequence in different ethnic populations has been revealed through MPS analysis, but the Korean population not only has limited MPS data for the entire mtGenome, the existing data is mainly focused on the control region. In this study, the complete mtGenome data for 186 Koreans, obtained using Ion Torrent Personal Genome Machine (PGM) technology and retrieved from rather common mtDNA haplogroups based on the control region sequence, are described. The results showed that 24 haplogroups, determined with hypervariable regions only, branched into 47 subhaplogroups, and point heteroplasmy was more frequent in the coding regions. In addition, sequence variations in the coding regions observed in this study were compared with those presented in other reports on different populations, and there were similar features observed in the sequence variants for the predominant haplogroups among East Asian populations, such as Haplogroup D and macrohaplogroups M9, G, and D. This study is expected to be the trigger for the development of Korean specific mtGenome data followed by numerous future studies.

Genomic profiling in a homogeneous molecular subtype of non‑small cell lung cancer: An effort to explore new drug targets.

BACKGROUND: Patients’ who are positive for kinase domain activating mutations in epidermal growth factor receptor (EGFR) gene, constitute 30–40% of non‑small cell lung cancer (NSCLC), and are suitable candidates for Tyrosine Kinase Inhibitor based targeted/personalized therapy. In EGFR non‑mutated subset, 8–10% that show molecular abnormalities such as EML4‑ALK, ROS1‑ALK, KIP4‑ALK, may also derive the benefit of targeted therapy. However, 40% of NSCLC belong to a grey zone of tumours that are negative for the clinically approved biomarkers for personalized therapy. This pilot study aims to identify and classify molecular subtypes of this group to address the un‑met need for new drug targets in this category. Here we screened for known/novel oncogenic driver mutations using a 46 gene Ampliseq Panel V1.0 that includes Ser/Thr/ Tyr kinases, transcription factors and tumor suppressors. METHODS: NSCLC with tumor burden of at least 40% on histopathology were screened for 29 somatic mutations in the EGFR kinase domain by real‑time polymerase chain reaction methods. 20 cases which were EGFR non‑mutated for TK domain mutations were included in this study. DNA Quality was verified from each of the 20 cases by fluorimeter, pooled and subjected to targeted re‑sequencing in the Ion Torrent platform. Torrent Suite software was used for next generation sequencing raw data processing and variant calling. RESULTS: The clinical relevance and pathological role of all the mutations/variants that include SNPs and Indels was assessed using polyphen‑2/SIFT/PROVEAN/mutation assessor structure function prediction programs. There were 10 pathogenic mutations in six different oncogenes for which annotation was available in the COSMIC database; C420R mutation in PIK3CA, Q472H mutation in vascular endothelial growth factor receptor 2 (VEGFR2) (KDR), C630W and C634R in RET, K367M mutation in fibroblast growth factor receptor 2 (FGFR2), G12C in KRAS and 4 pathogenic mutations in TP53 in the DNA binding domain (E285K, R213L, R175H, V173G). CONCLUSION: Results suggest, a potential role for PIK3CA, VEGFR2, RET and FGFR2 as therapeutic targets in EGFR non‑mutated NSCLC that requires further clinical validation.

Whole-genome sequences of influenza A(H3N2) viruses isolated from Brazilian patients with mild illness during the 2014 season

The influenza A(H3N2) virus has circulated worldwide for almost five decades and is the dominant subtype in most seasonal influenza epidemics, as occurred in the 2014 season in South America. In this study we evaluate five whole genome sequences of influenza A(H3N2) viruses detected in patients with mild illness collected from January-March 2014. To sequence the genomes, a new generation sequencing (NGS) protocol was performed using the Ion Torrent PGM platform. In addition to analysing the common genes, haemagglutinin, neuraminidase and matrix, our work also comprised internal genes. This was the first report of a whole genome analysis with Brazilian influenza A(H3N2) samples. Considerable amino acid variability was encountered in all gene segments, demonstrating the importance of studying the internal genes. NGS of whole genomes in this study will facilitate deeper virus characterisation, contributing to the improvement of influenza strain surveillance in Brazil.

Application of Ion Torrent PGMTM System in Detection of Fetal DNA in Maternal Plasma / 法医学杂志

Objective To explore the feasibility of detecting of Y-STR of fetal DNA in m aternal plasm a using Ion Torrent PGMTM System . Methods A total of 16 fetal DNA sam ples from m aternal plasm as (8 cases from 38 w eeks gestational age and 8 ones from 12 w eeks) w ere prepared and a m ultiplex assay w ith 7 STR loci (DYS390,DYS391,DYS393,DYS438,DYS437,DYS456,DYS635) w as designed for m ul-tiplex-PC R am plification. U sing Ion Torrent PGMTM System , the results of Y-STR sequences and capillary electrophoresis w ere obtained and com pared. Results Y-STR specific alleles w ere detected in the m ater-nal plasm a of all the pregnant w om en having m ale babies of second and third trim ester, w hich w ere higher than that detected by capillary electrophoresis. C onsistent Y-STR genotypes w ere observed betw een fetal DNA from m aternal plasm a and genom ic DNA from the new born babies. Conclusion B ased on Ion Torrent PGMTM System , the prenatal Y-STR detection m ethod m ay provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.

Performance Comparison of Benchtop Next-generation Sequencing Systems

With fast development and wide applications of next generation sequencing (NGS), genomic sequence information is within reach to various research fields. Three benchtop NGS instruments are now available. The 454 GS Junior (Roche), Ion PGM (Life Technologies) and MiSeq (Illumina) are laser-printer sized and offer modest set-up and running costs. By reviewing 2 studies that compared the performance of these instruments, the major characteristics of each benchtop platforms are compared to enable direct comparisons. The 454 GS Junior generated the longest reads and most contiguous assemblies but had the lowest throughput. The Ion Torrent PGM had the highest throughput and fastest run time. The MiSeq had the highest throughput per run and lowest error rates. The Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors. Although all the platforms allow multiplexing of samples, details of experimental design, library preparation and data analysis may constrain the options. The features of the platforms provide opportunities both to conduct groundbreaking studies and to waste money. Thus, careful considerations should be made before purchasing or using any of them.