Ion Torrent Next-generation Sequencing Research Articles

P114 Validation of high resolution HLA typing using NXTypeTM Ion-Torrent next generation sequencing. The University of Miami experience

Aim HLA typing using next generation sequencing (NGS) takes the Sanger based sequencing HLA typing to a higher dimension. The technology permits multiplexing of several target regions and various samples in a single run. The advantages of this technology include high throughput, improved DNA sequencing accuracy and faster data analysis. Our aim was validate the NXTypeTM Ion-Torrent NGS HLA typing procedure to implement it in our clinical laboratory. Methods 214 samples were analyzed with NXType™ Ion-Torrent NGS and compared to high resolution typing obtained by Sanger sequencing. HLA Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) were amplified with NXType™ Class I and Class II primers set, respectively. Amplicons were fragmented with Ion Shear™ Plus Reagents Kit to sizes from 350 to 950bp. Libraries were enriched in OneTouch ES and run in an Ion PGM™. Data was analyzed with HLATypeStream (v1.0.0.86) software. Results We obtained good coverage of the target regions when multiplexing 24 samples per run using a 318™ v2 chip. Of the 1070 alleles analyzed (214 samples), 143 were homozygous and 927 heterozygous. The concordance between Sanger sequencing results and NGS was 99.2%, 99.6%, 99.8%, 100% and 99.8% for loci A, B, C, DRB1 and DQB1, respectively. The number of different alleles identified for loci A, B, C, DRB1 and DQB1 were 40, 69, 36, 42, and 18, respectively. Our results revealed that 269 NMDP codes reported by Sanger sequencing were resolved by NGS, except 3 codes in DRB1 because of mismatches present in exons 1 or 4, which are not targeted by NXType™ Class II primers. We found two new alleles in exons (A ∗ 24, exon 1 and C ∗ 01, exon 2) and variations in intronic regions of 46 alleles. All null alleles A ∗ 68:11N (10), B ∗ 15:01N (11) and C ∗ 04:09N/B ∗ 44:03 (3) seen by Sanger were resolved by NGS. Conclusions The NGS is a reliable and accurate method to determine HLA typing of a considerable number of samples relatively fast, solving most allele ambiguities (98.7%) without the need of alternative tests. We confirmed that NXTypeTM Ion-Torrent NGS is a suitable and quick method for routine use in clinical laboratories to obtain complete ultra-high-resolution HLA typing that ultimately will improve on current HLA matching for transplant.

Erratum for Salipante et al., Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling.

Volume 80, no. 24, p. 7583–7591, 2014. Supplemental material. Table S8 included incorrect data in the “Error type,” “Platform,” and “Read orientation” columns corresponding to the per-base error rates in the leftmost column. Revised supplemental material is posted at <http://aem.asm.

Metagenome changes in the biogas producing community during anaerobic digestion of rice straw

The present investigation was undertaken to study the microbial community succession in a sour and healthy digester. Ion torrent next-generation sequencing (NGS)-based metagenomic approach indicated abundance of hydrolytic bacteria and exclusion of methanogens and syntrophic bacteria in sour digester. Functional gene analysis revealed higher abundance of enzymes involved in acidogenesis and lower abundance of enzymes associated with methanogenesis like Methyl coenzyme M-reductase, F420 dependent reductase and Formylmethanofuran dehydrogenase in sour digester. Increased abundance of methanogens (Methanomicrobia) and genes involved in methanogenesis was observed in the restored/healthy digester highlighting revival of pH sensitive methanogenic community.

Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: validation analysis of DMD mutations.

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited neuromuscular disease. The genetic diagnosis is not easily made because of the large size of the dystrophin gene, complex mutational spectrum and high number of tests patients undergo for diagnosis. Multiplex ligation-dependent probe amplification (MLPA) has been used as the initial diagnostic test of choice. Although MLPA can diagnose 70% of DMD/BMD patients having deletions/duplications, the remaining 30% of patients with small mutations require further analysis, such as Sanger sequencing. We applied a high-throughput method using Ion Torrent next-generation sequencing technology and diagnosed 92% of patients with DMD/BMD in a single analysis. We designed a multiplex primer pool for DMD and sequenced 67 cases having different mutations: 37 with deletions/duplications and 30 with small mutations or short insertions/deletions in DMD, using an Ion PGM sequencer. The results were compared with those from MLPA or Sanger sequencing. All deletions were detected. In contrast, 50% of duplications were correctly identified compared with the MLPA method. Small insertions in consecutive bases could not be detected. We estimated that Ion Torrent sequencing could diagnose ~92% of DMD/BMD patients according to the mutational spectrum of our cohort. Our results clearly indicate that this method is suitable for routine clinical practice providing novel insights into comprehensive genetic information for future molecular therapy.

Abstract P5-06-05: MED12 exon 2 mutations in phyllodes tumors of the breast

Abstract Exon 2 of MED12, a subunit of the transcriptional mediator complex, has been frequently mutated in uterine leiomyomas and breast fibroadenomas; however, it has been rarely mutated in other tumors. Although the mutations were also found in uterine leiomyosarcomas, the frequency was significantly lower than in uterine leiomyomas. Here, we examined the MED12 mutation in phyllodes tumors, another biphasic tumor with epithelial and stromal components related to breast fibroadenomas. Mutations in MED12 exon 2 were analyzed in nine fibroadenomas and eleven phyllodes tumors via Sanger sequencing. A panel of cancer- and sarcoma-related genes was also analyzed using Ion Torrent next-generation sequencing. Six mutations in fibroadenomas, including those previously reported (6/9, 67%), and five mutations in phyllodes tumors (5/11, 45%) were observed. Three mutations in the phyllodes tumors were missense mutations at Gly44, which is common in uterine leiomyomas and breast fibroadenomas. In addition, two deletion mutations (in-frame c.133_144del12 and loss of splice acceptor c.100-68_137del106) were observed in the phyllodes tumors. No other recurrent mutation was observed with next-generation sequencing. Frequent mutations in MED12 exon 2 in the phyllodes tumors suggest that it may share genetic etiology with uterine leiomyoma, a subgroup of uterine leiomyosarcomas and breast fibroadenoma. Citation Format: Nagasawa S, Ohta T, Tsugawa K. MED12 exon 2 mutations in phyllodes tumors of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-05.

Strategies for complete mitochondrial genome sequencing on Ion Torrent PGM™ platform in forensic sciences

Next generation sequencing (NGS) is a time saving and cost-efficient method to detect the complete mitochondrial genome (mtGenome) compared to Sanger sequencing. In this study we focused on developing strategies for mtGenome sequencing on the Ion Torrent PGM™ platform and NGS data analysis. With our experience, 4, 15 and 30 samples could be loaded onto Ion 314™, Ion 316™ and Ion 318™ chips respectively at a pooling concentration of 26pM, achieving to sufficient average coverage of ≥1500×and well strand balance of 1.05. Data processing software is essential to NGS mega data analysis. The in-house Perl scripts were developed for primary data analysis to screen out uncertain positions and samples from variant call format (VCF) reports and for pedigree study to perform pairwise comparisons. The Integrative Genomic Viewer (IGV) and the NextGENe software were introduced to secondary data analysis. The mthap and EMMA were employed for haplogroup assignment. The dataset was reviewed and approved by the EMPOP as the final version, which showed 2.66% error rate generated from the Torrent Variant Caller (TVC). Across the mtGenome, 4022 variants were found at 725 nucleotide positions, where ratio of transitions to transversions was estimated at 20.89:1 and 22.18% of variants was concentrated at hypervariable segments I and II (HVS-I and HVS-II). Totally, 107 complete mtGenome haplotypes were observed from 107 Northern Chinese Han and assigned to 88 haplogroups. The random match probability (RMP) of complete mtGenome was calculated as 0.009345794, decreasing 26.19% by comparison to that of HVS-I only, and the haplotype diversity (HD) was evaluated as 1, increasing 0.33% by comparison to that of HVS-I only. Principal component analysis (PCA) showed that our population was clustered to East and Southeast Asians. The strategies in this study are suitable for complete mtGenome sequencing on Ion Torrent PGM™ platform and Northern Chinese Han (EMP00670) is the first complete mtGenome dataset contributed to the EMPOP from East Asian.

Erratum to Performance comparison of illumina and ion torrent next-generation sequencing platforms for 16s rrna-based bacterial community profiling [Applied and Environmental Microbiology 80, 24, (2014), 7583-7591

Erratum to Performance comparison of illumina and ion torrent next-generation sequencing platforms for 16s rrna-based bacterial community profiling [Applied and Environmental Microbiology 80, 24, (2014), 7583-7591

Previously Unidentified Single Nucleotide Polymorphisms in HIV/AIDS Cases Associate with Clinical Parameters and Disease Progression.

The genetic background of an individual plays an important role in the progression of HIV infection to AIDS. Identifying previously unknown or uncharacterized single nucleotide polymorphisms (SNPs) that associate with disease progression may reveal important therapeutic targets and provide a greater understanding of disease pathogenesis. In the present study, we employed ultra-high multiplex PCR on an Ion Torrent next-generation sequencing platform to sequence 23 innate immune genes from 94 individuals with HIV/AIDS. This data was used to identify potential associations of SNPs with clinical parameters and disease progression. SNPs that associated with an increased viral load were identified in the genes for the interleukin 15 receptor (IL15RA), toll-like receptor 7 (TLR7), tripartite motif-containing protein 5 (TRIM5), and two killer-cell immunoglobulin-like receptors (KIR2DL1 and KIR2DL3). Additionally, SNPs that associated with progression from HIV infection to AIDS were identified in two 2′-5′-oligoadenylate synthetase genes (OAS2 and OAS3). In contrast, other SNPs identified in OAS2 and OAS3 genes, as well as in the TRIM5 and KIR2DS4 genes, were associated with a slower progression of disease. Taken together, our data demonstrates the utility of ultra-high multiplex PCR in identifying polymorphisms of potential clinical significance and further,identifies SNPs that may play a role in HIV pathogenesis.

Improved Detection by Next-Generation Sequencing of Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% (n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% (n = 88), 83% (n = 88), and 89% (n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Next-generation sequencing for molecular diagnosis of lung adenocarcinoma specimens obtained by fine needle aspiration cytology.

Identification of multi-gene variations has led to the development of new targeted therapies in lung adenocarcinoma patients, and identification of an appropriate patient population with a reliable screening method is the key to the overall success of tumor targeted therapies. In this study, we used the Ion Torrent next-generation sequencing (NGS) technique to screen for mutations in 89 cases of lung adenocarcinoma metastatic lymph node specimens obtained by fine-needle aspiration cytology (FNAC). Of the 89 specimens, 30 (34%) were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations. Seven (8%) samples harbored KRAS mutations, and three (3%) samples had BRAF mutations involving exon 11 (G469A) and exon 15 (V600E). Eight (9%) samples harbored PIK3CA mutations. One (1%) sample had a HRAS G12C mutation. Thirty-two (36%) samples (36%) harbored TP53 mutations. Other genes including APC, ATM, MET, PTPN11, GNAS, HRAS, RB1, SMAD4 and STK11 were found each in one case. Our study has demonstrated that NGS using the Ion Torrent technology is a useful tool for gene mutation screening in lung adenocarcinoma metastatic lymph node specimens obtained by FNAC, and may promote the development of new targeted therapies in lung adenocarcinoma patients.

MED12 exon 2 mutations in phyllodes tumors of the breast.

Exon 2 of MED12, a subunit of the transcriptional mediator complex, has been frequently mutated in uterine leiomyomas and breast fibroadenomas; however, it has been rarely mutated in other tumors. Although the mutations were also found in uterine leiomyosarcomas, the frequency was significantly lower than in uterine leiomyomas. Here, we examined the MED12 mutation in phyllodes tumors, another biphasic tumor with epithelial and stromal components related to breast fibroadenomas. Mutations in MED12 exon 2 were analyzed in nine fibroadenomas and eleven phyllodes tumors via Sanger sequencing. A panel of cancer- and sarcoma-related genes was also analyzed using Ion Torrent next-generation sequencing. Six mutations in fibroadenomas, including those previously reported (6/9, 67%), and five mutations in phyllodes tumors (5/11, 45%) were observed. Three mutations in the phyllodes tumors were missense mutations at Gly44, which is common in uterine leiomyomas and breast fibroadenomas. In addition, two deletion mutations (in-frame c.133_144del12 and loss of splice acceptor c.100-68_137del106) were observed in the phyllodes tumors. No other recurrent mutation was observed with next-generation sequencing. Frequent mutations in MED12 exon 2 in the phyllodes tumors suggest that it may share genetic etiology with uterine leiomyoma, a subgroup of uterine leiomyosarcomas and breast fibroadenoma.

Next-Generation Sequencing-Based Genetic Testing For Familial Partial Lipodystrophy

ABSTRACT Objective: Familial partial lipodystrophy (FPL) of the Dunnigan type (FPLD) is an autosomal dominant condition characterized by fat loss in the limbs and trunk, fat accumulation in the head and neck, and early onset diabetes mellitus. Here we describe the establishment and utilization of next-generation sequencing (NGS)-based genetic testing for FPLD. Methods: We describe NGS-based mutational analysis of the lamin A/C (LMNA) gene, followed by confirmation through Sanger sequencing. Results: We report a patient and her mother with accumulation of fat in the neck and face and loss of fat in the limbs and trunk typical of FPLD2, with young onset diabetes mellitus without ketoacidosis. Both subjects had elevated homeostasis model assessment estimated insulin resistance (HOMA-IR) values and serum triglyceride levels, indicating insulin resistance. Dual energy X-ray absorptiometry confirmed typical fat redistribution. NGS-based mutational analysis of the LMNA gene in these patients revealed a hot spot missense mutation (c.1444C>T, p.Arg482Trp) that was further confirmed by Sanger sequencing. Conclusion: A high index of clinical suspicion is essential to make an accurate clinical diagnosis in subjects with diabetes at an early age. FPLD should be considered in the differential diagnosis of monogenic diabetes with lipodystrophy. Ion torrent NGS offers flexibility and a multiplexing option that provides a robust and inexpensive platform for screening single-gene disorders. Abbreviations: FPL familial partial lipodystrophy FPLD familial partial lipodystrophy of the Dunnigan type NGS next-generation sequencing

Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients

AimsTo evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to...

Abstract 1523: Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel

Abstract Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.80%), KIT (33.60%), and PIK3CA (41.60%) genes. The mutations in MLH1 gene were frequently detected in the exons 2, while MET gene was frequently mutated in the exon 12. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy. Citation Format: Zhi Xu, Zhibin Hu, Xinying Huo, Chuanning Tang, Si-Yi Chen, Jinfei Chen. Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2014-1523

Development of ten microsatellite loci in the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822

A suite of tetra-nucleotide microsatellite loci were developed for the invasive giant African land snail, Achatina (=Lissachatina) fulica Bowdich, 1822, from Ion Torrent next-generation sequencing data. Ten of the 96 primer sets tested amplified consistently in 30 snails from Miami, Florida, plus 12 individuals representative of their native East Africa, Indian and Pacific Ocean regions. The loci displayed moderate levels of allelic diversity (average 5.6 alleles/locus) and heterozygosity (average 42 %). Levels of genetic diversity were sufficient to produce unique multi-locus genotypes and detect phylogeographic structuring among regional samples. The invasive A. fulica can cause extensive damage to important food crops and natural resources, including native flora and fauna. The loci characterized here will be useful for determining the origins and tracking the spread of invasions, detecting fine-scale spatial structuring and estimating demographic parameters.

Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002.

Tumor somatic mutation analysis is part of the standard management of metastatic lung cancer. However, physicians often have to deal with small biopsies and consequently with challenging mutation testing. Circulating free DNA (cfDNA) is a promising tool for accessing the tumor genome as a liquid biopsy. Here, we evaluated next-generation sequencing (NGS) on cfDNA samples obtained from a consecutive series of patients for the screening of a range of clinically relevant mutations. A total of 107 plasma samples were collected from the BioCAST/IFCT-1002 lung cancer study (never-smokers cohort). Matched tumor DNA (tDNA) was obtained for 68 cases. Multiplex PCR-based assays were designed to target specific coding regions in EGFR, KRAS, BRAF, ERBB2, and PI3KCA genes, and amplicon sequencing was performed at deep coverage on the cfDNA/tDNA pairs using the NGS IonTorrent Personal Genome Machine Platform. CfDNA concentration in plasma was significantly associated with both stage and number of metastatic sites. In tDNA, 50 mutations (36 EGFR, 5 ERBB2, 4 KRAS, 3 BRAF, and 2 PIK3CA) were identified, of which 26 were detected in cfDNA. Sensitivity of the test was 58% (95% confidence interval, 43%-71%) and the estimated specificity was 87% (62%-96%). These data demonstrate the feasibility and potential utility of mutation screening in cfDNA using IonTorrent NGS for the detection of a range of tumor biomarkers in patients with metastatic lung cancer.

Isolation and characterisation of 18 polymorphic microsatellite loci for black marlin (Istiompax indica) and their utility for Pacific billfish species

We present the isolation and characterisation of 18 polymorphic microsatellite loci with perfect 3 and 4-bp repeats for the black marlin Istiompax indica using Ion Torrent next-generation sequencing techniques. Usefulness of these markers for genetic studies was assessed by screening 16 specimens collected throughout the Indo-Pacific basin for this broadly distributed and exploited species. A total of 30 black marlin from the West Coral Sea were genotyped producing between 2 and 20 alleles per locus (mean 5.27) with expected heterozygosity (HE) ranging from 0.095 to 0.843. The genotypic distribution of all loci conformed to Hardy–Weinberg expectations with no evidence of null alleles. Evaluations of cross-species amplification of these markers on five additional Pacific billfish species indicates the utility of these markers for genetic studies of other Istiophorids.

Frequent KIT mutations in human gastrointestinal stromal tumors.

Identifying gene mutations in individual tumors is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumors (GIST) are stromal or mesenchymal subepithelial neoplasms affecting the gastrointestinal tract and frequently contain activating gene mutations in either KIT or platelet-derived growth factor A (PDGFRA). Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival in patients with GIST. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. Utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes using DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 121 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in the KIT gene. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. This may provide a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy.

A meta-analysis of microRNAs with prognostic significance in pancreatic ductal adenocarcinoma

s / Pancreatolog S84 Background: The circulating tumour cells (CTCs) responsible for metastasis in pancreatic cancer (PDAC) are heterogeneous; tailoring therapy based on their molecular profile could permit personalised therapy and improve survival. Aims: Optimise whole blood enrichment to allow next generation sequencing (NGS) of CTCs in PDAC. Materials & methods: An experimental sample, 10 Panc-1 cells spiked into 7.5mls of whole blood, and a clinical sample, 7.5mls of whole blood from a patient with advanced PDAC were used. Streptavidin coated magnetic beads were chemically bonded with CD45 antibodies specific to leucocytes and thoroughly mix with the samples. Leukocytes were removed with a magnet. DNA was isolated from the supernatant using Roche Magnupure. Libraries were diluted to 10 genomes and multiple independent barcoded assays were run per analysis. Barcoded primers for exons 5,6,7 and 8 of Tp53 were used for PCR amplification. Deep sequencing of Tp53 was performed with ion torrent NGS. 10% mutation sequence in one of 100 barcodes was taken as evidence of 0.1% mutation frequency. The limiting dilution removes the possibility of low level PCR error being confused with genuine mutation. Results: The known p.R273H mutation in Panc-1 cells was identified at a frequency 2% in the spiked sample. Amutation p.E271Vwas identified at a frequency 3.4% in the processed sample but was undetectable (<0.01%) in the controls and unprocessed sample. Conclusion: We have optimised a method of CTC enrichment sufficient to identify mutations in Tp53 in both experimental and clinical blood samples. This paves the way for personalised adjuvant chemotherapy in PDAC.