Personal Genome MachineTM Research Articles

Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material.

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel's ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available OncomineTM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the OncomineTM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing.

Alternaria alternata Causing Disease on Sugar Beet (Beta vulgaris) Steckling in Arizona, USA

Sugar beet (Beta vulgaris. L) is an economically important crop which is contributing 55% of the total sugar in the USA. In April 2018, irregular dark brown symptoms were observed in sugar beet stecklings in Arizona where different sugar beet cultivars were grown for seed production (Figure 1). The symptoms covered approximately 5% on the sugar beet root surface. Symptomatic beet root tissue were excised from the junction of diseased and healthy tissue. Small pieces (5mm²) were surface sterilized with 10% sodium hypochlorite for 1min, rinsed thrice with sterile distilled water, air dried and transferred to clarified V8 (CV8), and Potato Dextrose Agar (PDA), and incubated at 24°C with a 12h photoperiod for 5 days. White-dark green velvety colony appeared on both media (Figure 2). Isolates were developed by the single spore isolation technique. Conidia were obclavate or oviod, two to four transverse septa, and pale brown, often in chains (4 to 8 conidia) and or solitary (Figure 3). The dimension of conidia varied from 20.21-40.15 x 7.50-14.12 μm [1- 3]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic DNA were extracted via Qiagen kit. A total of 40ng genomic DNA was used to generate a library using NEBNext® Fast DNA Fragmentation & Library Prep Set for Ion TorrentTM for sequencing in Ion TorrentTM Personal Genome MachineTM System.

Impact of Inoculation with Pseudomonas aestus CMAA 1215T on the Non-target Resident Bacterial Community in a Saline Rhizosphere Soil.

Plant growth reduction caused by osmotic stress, pathogens, and nutrient scarcity can be overcome by inoculation with plant growth-promoting rhizobacteria (PGPR). Knowing the effects of PGPR on the microbial community beyond those on plant growth can bring new options of soil microbiota management. The present study aimed to investigate the effect of inoculation with the newly described Pseudomonas aestus CMAA 1215T [a 1-aminocyclopropane-1-carboxylate (ACC) deaminase and glycine-betaine producer] on the rhizosphere bacterial community of Zea mays in natural (non-salinized) and saline soil. The bacterial community structure was assessed by sequencing the V6-V7 16S ribosomal RNA using the Ion Personal Genome Machine™. The non-metric multidimensional scaling (NMDS) of the OTU profile (ANOSIM P < 0.01) distinguishes all the treatments (with and without inoculation under saline and natural soils). Inoculated samples shared 1234 OTUs with non-inoculated soil. The most abundant classes in all samples were Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Acidobacteriia, Bacteroidia, Thermoleophilia, Verrucomicrobiae, Ktenodobacteria, and Bacilli. The inoculation, on the other hand, caused an increase in the abundance of the genera Bacillus, Bryobacter, Bradyrhizobium, "Candidatus Xiphinematobacter", and "Candidatus Udaeobacter" independent of soil salinization. "Candidatus Udaeobacter" has the largest Mean Decrease in Gini Values with higher abundance on inoculated salted soil. In addition, Pseudomonas inoculation reduced the abundance of Gammaproteobacteria and Phycisphaerae. Understanding how inoculation modifies the bacterial community is essential to manage the rhizospheric microbiome to create a multi-inoculant approach and to understand its effects on ecological function.

Pectobacterium carotovorum subsp. actinidiae associated with canker on pear trees in Brazil

In summer of 2016, cankers were observed on 8-year-old pear trees (Pyrus communis L.) cv. Yali grafted on the Caleriana rootstock, in a commercial orchard in Monte Castelo, SC. Eight hundred out of 1200 trees died and the rest of the orchard was eradicated. Affected plants showed cankers on trunks and twigs with exudation; the branches turned brown and slowly died. Bacterial colonies were isolated on King’s B medium from infected tissues. Pathogenicity, biochemical tests were conducted and some isolates selected for whole genome sequencing using Ion Personal Genome Machine™ (PGM™) System (ThermoFisher Scientific). PCR with Y1/Y2 and PcaF/PcaR primers, specific for Pectobacterium carotovorum and Pectobacterium carotovorum subsp. actinidiae, respectively, were also carried out. The section of inoculated apple and pear branches showed longitudinal darkening, with wilt and leaf blight. Shoots of kiwifruit Actinidia deliciosa cv. Hayward inoculated with the bacterium showed no symptoms. The bacterium was re-isolated from all symptomatic tissues, satisfying Koch’s postulates. Selected isolates were gram-negative, facultative anaerobic, rod-shaped and exhibited a strong pectolytic activity in potato and carrot disks, and showed similar biochemical profile using a BIOLOG GEN III MicroPlate. The pathogen of the symptoms observed on pear trees in Brazil show highest genome (87.1%; DDH analysis) and proteome identity (99.8%; Patric analysis) with P. carotovorum subsp. actinidiae. A product of the expected size was obtained in both specific PCR (Y1/Y2 (434 bp) and PcaF/PcaR (343 bp) primer pairs). Results from the present study clearly show that P. carotovorum subsp. actinidiae was associated with canker on pear trees in Santa Catarina, Brazil.

Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the “same donor” or from a “first degree relative” was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21–26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18–23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10−13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping.

Searching the undetected mtDNA variants in forensic MPS data

The efficiency of MPS in forensic mtDNA analysis has been thoroughly proven, although a reliable and well established data evaluation still remains a critical point. Numerous bioinformatics tools have been developed, but most of them require specific operating systems and high costs, while free open-source programs with user-friendly interfaces are few. In this study, 43 full mtGenomes were sequenced using the Ion Personal Genome Machine™ (PGM™) System and analyzed utilizing the plug-in Variant Caller (TVC) of the Ion Torrent Software Suite and the mtDNA-Server (mDS), a free web-based mitochondrial analysis tool for MPS data. The outcomes of these two different analysis tools were compared to variants noted after manual inspection of the aligned reads performed using Integrative Genomics Viewer (IGV). The comparison highlighted the presence of thirty-nine discordant variant calls, which were resolved by Sanger sequencing that confirmed the presence of all variants, except for 7 deletions. The combined adoption of IGV and Sanger type sequencing confirmatory steps, in addition of TVC and mDS analysis, resulted in a more accurate variants assignment with the detection of 32 additional true polymorphisms, which were noted in the final dataset. Regarding the heteroplasmy issue, out of a total of thirty heteroplasmic variants, twenty-eight were detected by the TVC, while the mDS detected twenty-two.Overall, none of the used bioinformatics tools were the perfect choice and a secondary analysis with an expert's opinion in complete mtGenome MPS data evaluation is still required in forensic genetic analysis.

Somatic mutations in planar cell polarity genes in neural tissue from human fetuses with neural tube defects.

Extensive studies that have sought causative mutation(s) for neural tube defects (NTDs) have yielded limited positive findings to date. One possible reason for this is that many studies have been confined to analyses of germline mutations and so may have missed other, non-germline mutations in NTD cases. We hypothesize that somatic mutations of planar polarity pathway (PCP) genes may play a role in the development of NTDs. Torrent™ Personal Genome Machine™ (PGM) sequencing was designed for selected PCP genes in paired DNA samples extracted from the tissues of lesion sites and umbilical cord from 48 cases. Sanger sequencing was used to validate the detected mutations. The source and distribution of the validated mutations in tissues from different germ layers were investigated. Subcellular location, western blotting, and luciferase assays were performed to better understand the effects of the mutations on protein localization, protein level, and pathway signaling. ix somatic mutations were identified and validated, which showed diverse distributions in different tissues. Three somatic mutations were novel/rare: CELSR1 p.Gln2125His, FZD6 p.Gln88Glu, and VANGL1 p.Arg374His. FZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus, and disrupted the colocalization of CELSR1 and FZD6. This mutation affected non-canonical WNT signaling in luciferase assays. VANGL1 p.Arg374His impaired the co-localization of CELSR1 and VANGL1, increased the protein levels of VANGL1, and influenced cell migration. In all, 7/48 (14.5%) of the studied NTD cases contained somatic PCP mutations. Somatic mutations in PCP genes (e.g., FZD6 and VANGL1) are associated with human NTDs, and they may occur in different stages and regions during embryonic development, resulting in a varied distribution in fetal tissues/organs.

Draft genome sequence of Pseudoalteromonas piscicida strain 36Y_RITHPW, a hypersaline seawater isolate from the south coast of Sonora, Mexico.

This study reports the draft genome sequence of Pseudoalteromonas piscicida strain 36Y_RITHPW, a marine Gammaproteobacteria that synthesises bioactive compounds with antagonistic activity against Vibrio parahaemolyticus, a multidrug-resistant strain that is the causative agent of acute hepatopancreatic necrosis disease (AHPND), reported in shrimp farm outbreaks from Asia to Mexico with mortality rates of 80-100%. The genome of P. piscicida 36Y_RITHPW was sequenced with an Ion Torrent™ Personal Genome Machine™ (PGM) platform. A total of 606805 reads were constructed for a 308.48Mbp and 33.5×coverage. A high-quality draft assembly and ordering of contigs was obtained with Mauve. The annotation was obtained with RAST and antiSMASH. The genome size consists of 5.15Mbp, with a total of 4548 genes, 4217 protein-coding sequences and a GC content of 43.3%. Several resistance genes as well as other genes involved in the production of bacteriocins and ribosomally synthesised antibacterial peptides are also present. Mining of this draft genome provides valuable information to explain the antagonistic capacity of P. piscicida 36Y_RITHPW, a useful strain as a potential probiotic in shrimp aquaculture against pathogenic V. parahaemolyticus.

First genome report on novel sequence types of Neisseria meningitidis: ST12777 and ST12778.

Neisseria meningitidis is an important causative agent of meningitis and/or sepsis with high morbidity and mortality. Baseline genome data on N. meningitidis, especially from developing countries such as India, are lacking. This study aimed to investigate the whole genome sequences of N. meningitidis isolates from a tertiary care centre in India. Whole-genome sequencing was performed using an Ion Torrent™ Personal Genome Machine™ (PGM) with 400-bp chemistry. Data were assembled de novo using SPAdes Genome Assembler v.5.0.0.0. Sequence annotation was performed through PATRIC, RAST and the NCBI PGAAP server. Downstream analysis of the isolates was performed using the Center for Genomic Epidemiology databases for antimicrobial resistance genes and sequence types. Virulence factors and CRISPR were analysed using the PubMLST database and CRISPRFinder, respectively. This study reports the whole genome shotgun sequences of eight N. meningitidis isolates from bloodstream infections. The genome data revealed two novel sequence types (ST12777 and ST12778), along with ST11, ST437 and ST6928. The virulence profile of the isolates matched their sequence types. All isolates were negative for plasmid-mediated resistance genes. To the best of our knowledge, this is the first report of ST11 and ST437 N. meningitidis isolates in India along with two novel sequence types (ST12777 and ST12778). These results indicate that the sequence types circulating in India are diverse and require continuous monitoring. Further studies strengthening the genome data on N. meningitidis are required to understand the prevalence, spread, exact resistance and virulence mechanisms along with serotypes.

Unravelling the composition of soil belowground microbial community before sowing transgenic cotton

Soils harbour enormously diverse bacterial communities that interact specifically with plants generating beneficial interactions between them. This study was the first approach to assess bacterial communities before sowing with three cotton genotypes, including both transgenic and conventional ones. The structure of bacterial communities was identified using the next generation sequencing analysis, ion torrent PGM (Personal Genome Machine™) sequencer technology, based on the V2–V3 16S rRNA gene region. Quantitative insights into microbial ecology pipeline were used to identify the structure and diversity of bacterial communities in bulk soil samples collected in the northeast of Mexico. Bulk soil textures and chemical properties, including most nutrients, were homogeneous in these bulk soil samples. Relative abundance analysis showed similar bacterial community structures. Dominant taxonomic phyla were Proteobacteria, Firmicutes, Acidobacteria, Actinobacteria, Gemmatimonadetes and Bacteroidetes, whereas the main families were Bacillaceae, Chitinophagaceae and Rhodospirillaceae with an abundance average of BS1 (bulk soil sample), BS2 and BS3 (24.85, 19.74 and 19.71%, respectively). Alpha diversity analysis showed a high diversity (Shannon and Simpson index) and a large value of the observed species found in bulk soils samples. These results allowed establishing the previous bacterial structural community in an unused soil before sowing it with a transgenic crop for the first time.

Molecular features underlying response to neoadjuvant gemcitabine, cisplatin, and sorafenib (S-GC) in muscle-invasive urothelial bladder carcinoma (MIBC): Insights for trials of new agents combined with chemotherapy in the field.

e16018 Background: S-GC neoadjuvant chemotherapy (NACT) was active in a phase 2 trial in MIBC, showing a response rate (downstaging to pT &lt; 2) of 54.3% in 46 pts within a single-arm, phase 2 trial ( Necchi et al, GU ASCO 2017). The addition of S to GC may help investigators unravelling key molecular features underlying response in MIBC. Methods: Analyses were made on pre- and post-treatment TURB and cystectomy (Cy) specimens (10µm sections) and matched germline DNA of 29/46 pts. Analyses included measurements of ERCC1 (by immunohistochemistry, IHC) on TURB specimens, baseline circulating VEGF levels (ELISA), copy number variations (CNV, Affymetrix), and next generation sequencing (NGS, custom gene panels through the Ion AmpliSeq Library Kit2.0™ and the Ion Torrent Personal Genome Machine™ platform). Results: We analyzed TURB samples from 15 responders (R) and 4 non-R (NR). An additional 10 Cy samples were used for NR. Baseline VEGF levels were not associated with PFS or OS on univariable analyses (p = 0.943 and p = 0.669, respectively). Differences in R vs. NR pts were found according to CNVs and NGS. CN gains on regions of chromosome (chr) 18 and 20 and loss in chr 5 were found in 56-60% of R and in none of the NR. Gains on regions of chr 3 and losses in chr 1 and 20 were more frequent in the NR ( &gt; 50%) vs. R ( &lt; 20%). The median number of somatic mutations (mut) per sample was 3 (range, 0-19). Missense mut in DNA-repair genes ( POLK; FANCM; PRKDC; BLM; MSH2; MSH6; RAD50) were found in 63.6% R vs 16.7% NR, in RAS-RAF pathway genes ( CDKN2A; RAC1; KRAS; NF1) in 45.4% vs 7.1%, in chromatin-remodelling genes ( ARID1A; ARID2; KDM5C; SMARCA4; ATRX; KMT2C; DAXX; SETD2) in 54.5% vs 16.7%, and in HER-family genes in R only (36.4%). ERCC1 IHC expression was found in 55.6% R vs 93.3% NR (p = 0.047). Conclusions: We identified distinct molecular features of R vs NR to S-GC in MIBC. These features may be linked to a potentially-additive effect of S and CT. The identification of the targets of CNVs will further elucidate the underlying biology. These results may help developing personalized medicine in MIBC with new more potent targeted agents with CT. Clinical trial information: NCT01222676.

Massive parallel sequencing of human whole mitochondrial genomes with Ion Torrent technology: an optimized workflow for Anthropological and Population Genetics studies

Investigation of human mitochondrial DNA variation patterns and phylogeny has been extensively used in Anthropological and Population Genetics studies and sequencing the whole mitochondrial genome is progressively becoming the gold standard. Among the currently available massive parallel sequencing technologies, Ion Torrent™ semiconductor sequencing represents a promising approach for such studies. Nevertheless, an experimental protocol conceived to enable the achievement of both as high as possible yield and of the most homogeneous sequence coverage through the whole mitochondrial genome is still not available. The present work was thus aimed at improving the overall performance of whole mitochondrial genomes Ion Torrent™ sequencing, with special focus on the capability to obtain robust coverage and highly reliable variants calling. For this purpose, a series of cost-effective modifications in standard laboratory workflows was fine-tuned to optimize them for medium- and large-scale population studies. A total of 54 human samples were thus subjected to sequencing of the whole mitochondrial genome with the Ion Personal Genome Machine™ System in four distinct experiments and using Ion 314 chips. Seven of the selected samples were also characterized by means of conventional Sanger sequencing for the sake of comparison. Obtained results demonstrated that the implemented optimizations had definitely improved sequencing outputs in terms of both variants calling efficiency and coverage uniformity, enabling to setup an effective and accurate protocol for whole mitochondrial genome sequencing and a considerable reduction in experimental time consumption and sequencing costs.

Abstract B21: Targeted next-generation sequencing of hotspots in cancer genes in normal-tumor breast cancer patients of African ancestry reveals novel and known mutations

Abstract Introduction: Genomic profiling of primary tumors and their concomitant normal tissues has provided critical information about the mechanisms associated with carcinogenesis. Furthermore, while comprehensive analysis of breast tumors have yielded actionable findings, breast cancer (BCa) remains one of the most common cancers worldwide and BCa incidences and mortality rates vary by population reflecting health disparities resulting from socioeconomic and biologic factors. Given that African American (AA) BCa cases often display distinctly worse clinicopathological features, we hypothesize that interrogating tumors from AAs could significantly aid in understanding the drivers of BCa carcinogenesis specifically in this population. These findings would aid in the identification of significantly mutated genes leading to the development of interventions for improved drug targeting of tumors in these populations with African Ancestry that display an increase propensity for harboring these previously unknown somatic variants. Methods: For this study, DNA from 16 normal and tumor tissue pairs from the “African American Familial Breast Cancer Study” (AAFBC) were utilized. Using targeted next generation sequencing (NGS) technologies, libraries for the Ion AmpliSeq™Cancer Hotspot Panel v2 (CHPv2) panel were created using 20ng of DNA and each sample was assigned a unique barcode. The CHPv2 is a commercially available primer pool for targeted NGS that amplifies 207 regions within 50 oncogenes for a total of 31kb. The resulting libraries were quantified and pooled with unequal volumes to allow varying depth of coverage between the matched tumor and normal pairs. The samples were sequenced on the Ion Personal Genome Machine™ (PGM™) System with a 316v2 chip. Sequence data from the PGM was processed using the Ion Torrent Suite v4 software package. Single nucleotide variants (SNVs), multi-nucleotide polymorphisms (MNPs), insertions or deletions were identified using Torrent Variant Caller Plugin v.4.6.0.7. Results: Sequencing resulted in 3,241,580 total usable reads with an average length of 126bp. The average coverage was 667x for tumor tissues and 165x for matched normal tissues. There were 88 unique variants representing 528 total variants with an average of 16.5 variants per sample. Of these, there were 52 germline variants that were expressed by both the normal and tumor samples. Five of these variants displayed a loss of one allele resulting in one homozygous (FGFR3) and 4 heterozygous mutations (PDGFRA, STK11 and ERBB4). The remaining 36 variants were somatic mutations of which 20 were novel and 16 have been described previously. The coding consequences were 56% missense mutations, 28% synonymous mutations, 8% frameshifts and 6% in-frame deletions. The most common coding mutations found in at least 3 patients were in TP53, SMARCB1, CDKN2A, NOTCH1 and PIK3CA. Citation Format: Luisel Ricks-Santi, John McDonald, Cha'Tonya Brown, Latiffany Prince, Muneer Abbas, Georgia Dunston. Targeted next-generation sequencing of hotspots in cancer genes in normal-tumor breast cancer patients of African ancestry reveals novel and known mutations. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B21.

Abstract B23: Targeted exome sequencing for effectors involving the MAPK and PI3K pathways of cutaneous melanoma in the minority population: Identifying unique molecular signatures

Abstract Background: Melanoma is a global problem, as its incidence is rising faster than any other malignancy for both men and women. Since Caucasians are primarily affected by melanoma, most of the clinical effort and patient education is focused on this cohort. In minorities, melanoma is usually detected at a more advanced stage, distributed on sun-protected areas and associated with a worse survival. Previous studies have depicted the relevance of alterations in the MAPK and PI3K pathways; however, inclusion of a multi-ethnic population comprising of unique groups such as Native Hawaiians, Pacific Islanders, Filipinos, and other Asians has been limited. Methods: IRB approval was obtained for this retrospective analysis. From 2008 to 2013, 10 Asian/Pacific Islander patients histologically confirmed to have invasive conventional (non-acral) cutaneous melanoma were evaluated at a tertiary institution. Data including age, gender, tumor location, Breslow thickness, ulceration, and mitoses per mm squared were compiled. Using a cloud based in silica primer design software target genes was chosen. Multiplex primers were generated. Coverage was assessed using the University of California, Santa Cruz (UCSC) and National Center for Biotechnology Information (NCBI) genome browsers to ensure that known pathogenic single nucleotide polymorphisms were captured from the latest dbSNP genome build. Targets included AKT, BRAF, C-KIT, MEK, MTOR, NRAS, and PI3K. This pre-designed multiplexed panel contains genes across families representing driver mutations in signaling pathways. Next generation sequencing was performed using the Ion Torrent Personal Genome Machine™. Results: Our findings demonstrated a unique molecular distribution. Interestingly in our non-Caucasian cohort of conventional melanoma, PI3K mutations were found in 30% (normally 2-6% in Caucasians) of the biospecimens, which supports our hypothesis that ethnic minorities may have a distinct biological profile. Furthermore, 40% of this minority cohort had a c-KIT mutation which is atypical for non-acral melanoma; in addition 2 of these c-KIT mutations were novel and not classically associated with melanoma in Caucasians. What is also compelling is that 50% had simultaneous mutations, including 30% that had a concomitant PI3K mutation. Demographics of the multi-ethnic cohort included mean age of 57.5 years (range 19-80) and male gender (4/10; 40 %). Tumor location primarily involved the extremity (8/10; 80%); median Breslow thickness was 1.97 mm (range 0.77-16.0), and mean mitoses per mm squared was 5.2 (range 0-16). Conclusion: This is compelling data that may be applied to patient selection in future trials utilizing targeted agents and assist with gleaning insight into the mechanism of melanoma genesis. Molecular profiling diverse cohorts may improve the identification of novel molecular signatures germane to melanoma biology and therapy. Note: This abstract was not presented at the conference. Citation Format: Shane Y. Morita. Targeted exome sequencing for effectors involving the MAPK and PI3K pathways of cutaneous melanoma in the minority population: Identifying unique molecular signatures. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B23.

Targeted next generation sequencing application in cardiac channelopathies: Analysis of a cohort of autopsy-negative sudden unexplained deaths

Genetic testing for cardiac channelopathies in sudden unexplained death (SUD) has developed substantially over the last years. The Next Generation Sequencing (NGS) technology provides an unprecedented opportunity to screen for genetic variations underlying arrhythmogenic genes in a short period of time at a low cost.The present study aimed to perform genetic testing with NGS technologies on the Ion Torrent Personal Genome Machine™ (Ion PGM™) sequencer, in targeting a total of 23 genes reported to be associated with inherited cardiac channelopathies in order to identify the possible cause of death in a cohort of post-mortem cases.The molecular analyses focused on 16 cases of SUD, aged less than 35 years old. In all cases, the cause of death could not be determined after a rigorous autopsy associated with histopathological and toxicological analyses according to the guidelines of the Association for European Cardiovascular Pathology. DNA was extracted from fresh frozen tissue.An average of 200 variants was identified per case. However, after the prioritization process using a new scoring program (VaRank) and after the conjunction of clinical data and molecular findings, four “likely pathogenic” variants (including two undescribed variants), were identified in three cases (18.75%) of our cohort in the genes KCNH2, ANK2, SCN5A and RYR2. One case, who died during psychiatric hospitalization after administration of a QT prolonging drug, showed a double “likely pathogenic” variant in Long QT genes (ANK2 and SCN5A) which may have predisposed to drug-induced cardiac arrhythmias.Our study illustrates that the NGS approach based on AmpliSeq™ libraries and Ion Torrent PGM™ sequencing may be an efficient approach, integrated to post-mortem examination. Given the massive amount of information generated by NGS, a rigorous filtration strategy of variants coupled with multidisciplinary collaboration is crucial to determine the potential pathogenic role of identified variants in the cause of death.

Blind study evaluation illustrates utility of the Ion PGM™ system for use in human identity DNA typing.

AimTo perform a blind study to assess the capability of the Ion Personal Genome Machine™ (PGM) system to sequence forensically relevant genetic marker panels and to characterize unknown individuals for ancestry and possible relatedness.MethodsTwelve genomic samples were provided by a third party for blinded genetic analysis. For these 12 samples, the mitochondrial genome and three PGM™ panels containing human identity single nucleotide polymorphisms (SNPs), ancestry informative SNPs, and short tandem repeats (STRs) were sequenced on the PGM™ system and analyzed.ResultsAll four genetic systems were run and analyzed on the PGM™ system in a reasonably quick time frame. Completeness of genetic profiles, depth of coverage, strand balance, and allele balance were informative metrics that illustrated the quality and reliability of the data produced. SNP genotypes allowed for identification of sex, paternal lineage, and population ancestry. STR genotypes were shown to be in complete concordance with genotypes generated by standard capillary electrophoresis-based technologies. Variants in the mitochondrial genome data provided information on population background and maternal relationships.ConclusionAll results from analysis of the 12 genomic samples were consistent with sample information provided by the sample providers at the end of the blinded study. The relatively easy identification of intra-STR allele SNPs offered the potential for increased discrimination power. The promising nature of these results warrants full validation studies of this massively parallel sequencing technology and its further development for forensic data analysis.

Next‐generation sequencing: academic overkill or high‐resolution routine blood group genotyping?

Blood group genotyping (BGG) has been in routine clinical practice ever since the molecular determination of blood groups was achieved, during the early to mid 1990s. These early methods were dependent on allele‐specific PCR to detect simple single nucleotide polymorphisms (SNPs) responsible for blood group expression. As our knowledge regarding the molecular background of blood groups advanced, so did the numbers of SNPs requiring detection which necessitated the switch from allele‐specific PCR to array‐based technology, notably slide and bead‐based approaches. All these methods thus described are totally dependent on the predefined genetic basis of each SNP. Hybrid blood group genes (as found in RH, ABO and MNS systems) are difficult to define by all of these aforementioned techniques. With the arrival of cheap next‐generation sequencing (NGS) approaches in the past 5 years, we have conducted long‐range PCR (LR‐PCR) coupled with NGS determination of the major blood group genes. By analysis of these data using an Ion Torrent Personal Genome Machine™ (PGM™), it is readily apparent that NGS can highly effectively be applied with high resolution to blood grouping at costs no more than current array‐based platforms. However, the complexity of the data obtained need careful filtering for effective clinical utilization, but provides useful insight on the evolution of blood groups and their environmental impacts which will be of undoubted value as an academic exercise, but of minimal cost (if any) to the original testing.

Minimal Residual Disease Monitoring In t(8;21) Acute Myeloid Leukemia Based On RUNX1-RUNX1T1 Fusion Quantification On Genomic DNA

BackgroundAcute myeloid leukemia (AML) with t(8;21) chromosomal translocation, leading to the RUNX1-RUNX1T1 fusion, belong to the favorable risk AML subset. However, relapse incidence may reach 30-40% in these patients. Minimal residual disease monitoring (MRD) based on the quantification of RUNX1-RUNX1T1 fusion transcript by real-time quantitative PCR (RQ-PCR) has been reported to be an independent prognostic factor for the risk of relapse. The specificity of the RUNX1-RUNX1T1 fusion and the high sensitivity of RQ-PCR techniques have made RUNX1-RUNX1T1 an ideal marker to assess treatment response in t(8;21) AML. Undetectable MRD could mean either that tumor cells persist in a latent state without RNA expression or that MRD level is below the sensitivity threshold. Studies in chronic myeloid leukemia showed that BCR-ABL DNA was still detectable in patients in long-term complete molecular response with undetectable BCR-ABL fusion transcript. Using a similar approach, we investigated the use of RUNX1-RUNX1T1 DNA as a MRD marker in t(8;21) AML, instead of RUNX1-RUNX1T1 mRNA. This approach allows linking results directly to the amount of leukemic cells, since each leukemic cell contains one copy of the RUNX1-RUNX1T1 sequence, while the level of RUNX1-RUNX1T1 mRNA may vary from a patient to another. MethodsThis study focuses on 17 patients with t(8;21) AML included in the CBF-2006 trial and for whom frozen material was available for further molecular analysis. Bone marrow and blood samples were collected at AML diagnosis and during follow-up, as defined in the CBF-2006 trial. Eight patients relapsed during follow-up and 9 were still in complete remission at the end of the study. Interestingly, 3 patients relapsed with a previously undetectable MRD (in blood and bone marrow samples). First, we identified the breakpoints in the RUNX1 and RUNX1T1 genes for each patient using long-range PCR approaches, coupled with next-generation sequencing (NGS) on Personal Genome Machine™ (PGM). The stability of the RUNX1-RUNX1T1 rearrangement at relapse was checked by Sanger sequencing. Then, we performed quantification of RUNX1-RUNX1T1 DNA by RQ-PCR using Taqman technology. For each patient, a primer pair and a probe were designed using the patient's unique RUNX1-RUNX1T1 breakpoint sequence. The forward and reverse primers were located in RUNX1 and RUNX1T1 genes, respectively, and the probe was located at the RUNX1-RUNX1T1 junction. Calibration curves were established using 10-fold dilutions of the diagnostic DNA of each patient in normal control DNA. Results were given as a ratio of chimeric DNA amount in the follow-up sample to chimeric DNA amount at diagnosis. ResultsChromosomal breakpoints were located in RUNX1 intron 5 for all patients. RUNX1T1 breakpoints were located in intron 1b for 15 patients, and in intron 1a for 2 patients (Fig. 1). Quantification failed for 1 patient which was further leave up. Between 2 and 7 follow-up samples were studied for the other patients (median 4.5). DNA monitoring was strongly correlated with RNA monitoring (Fig. 2). Sensitivity threshold, determined by the lowest diagnostic sample dilution which gives a signal, was 10-5 for 7 patients, 10-4 for 6 patients, and only 5.10-4 for 3 patients. MRD was detectable in 31 samples and undetectable in 30 samples by both methods, whereas MRD was detectable only on RNA in 7 samples, probably because of a lack of sensitivity of the RQ-PCR assay. Interestingly, RUNX1T1-RUNX1 DNA was detected in 3 samples from 2 patients who relapsed and for whom RUNX1T1-RUNX1 transcript was undetectable, despite a good RNA quality. [Display omitted] [Display omitted] ConclusionsOverall, RUNX1-RUNX1T1 MRD levels on DNA and RNA were quite similar. The level of mRNA expression did not seem to change during follow-up when compared with the amount of DNA. MRD monitoring on genomic DNA is a useful method, but with sensitivity variations depending on the patient's breakpoint sequence and the efficiency of the RQ-PCR assay. DNA has potential advantages: it is more stable than RNA and a best substrate for collection, processing, transport and storage. Additionally, interpretation of the results is easier because it is closely related to the number of leukemic cells. However, this method greatly increases complexity, time of implementation, and cost of monitoring MRD, which limits its interest in routine practice. Disclosures:No relevant conflicts of interest to declare.

A Comprehensive Assay for CFTR Mutational Analysis Using Next-Generation Sequencing

Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.

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Marrow donor registries continue to evolve their processes and technology with the goal of reducing time required to find a match for a patient in need. Critical factors in achieving this goal include facilitating donor recruitment, collecting samples that meet requirements for the HLA typing technologies, and integrating cost efficiently into laboratory procedures. Non-invasive saliva sample collection with the Oragene family of products delivers a high quality and reliable solution for collection, stabilization and transportation of DNA. Saliva samples are currently being used as a reliable source of DNA for technologies such as SSO, SSP, SBT. Here we demonstrate in a comparison with blood that saliva collected using the Oragene family of products is a reliable sample for next generation sequencing. Saliva was collected using the Oragene collection device and then stored at room temperature until processing. Blood was collected into an EDTA tube and processed immediately by purifying on a silica column. Saliva samples were purified using PrepIT ® L2P from DNA Genotek. In this study we used the Raindance HLASeq™ research panel to tile amplicons across the entire HLA locus, which includes all Class I and Class II genes. The generated amplicons were then sequenced on the Ion Torrent™ Personal Genome Machine™ using the Ion 318™ chip. DNA extracted from saliva collected using Oragene was successfully amplified using the Raindance HLASeq panel and sequenced using the Ion Torrent PGM. The generated sequences were then compared between the paired samples and against results for the same donors assessed using traditional technologies. These results demonstrate a high concordance between paired blood and saliva samples. The results confirm that saliva collected using the Oragene family of products is an excellent source of DNA for the Raindance HLASeq panel and the high quality DNA is suitable for HLA typing by next generation sequencing. Iwasiow: DNA Genotek Inc.: Employee. Tayeb: DNA Genotek: Employee.