Ion Proton Platform Research Articles

Neuromuscular Choristoma: Report of Five Cases With CTNNB1 Sequencing.

Neuromuscular choristoma (NMC) are lesions of the peripheral nervous system characterized by an admixture of skeletal muscle fibers and nerves fascicles that are frequently associated with desmoid fibromatosis (DF). Mutations in CTNNB1, the gene for β-catenin protein, are common in DF and related to its pathogenesis. They are restricted to exon 3, with 3 point mutations: T41A, S45F, and S45P. To understand the pathogenesis of NMC, we tested CTNNB1 status in 5 cases of NMC whether or not they were associated with DF. The screening of mutations in CTNNB1 gene was based on amplicon deep sequencing using the ION Proton platform. Three patients had the S45F mutation; in 2 the mutation was common to both lesions and in one the DF was wild type while the NMC had the S45F mutation. One patient had a T41A mutation in the NMC and no associated DF. In the last patient, the DF lesion had a T41A mutation; there was no lesion with the S45P mutation. The presence of similar CTNNB1 mutations in NMC/DF-associated lesions and sporadic DF reinforces the relationship between both lesions and points to a common pathogenic mechanism.

Development and validation of an expanded targeted sequencing panel for non-invasive prenatal diagnosis of sporadic skeletal dysplasia

BackgroundSkeletal dysplasia (SD) is one of the most common inherited neonatal disorders worldwide, where the recurrent pathogenic mutations in the FGFR2, FGFR3, COL1A1, COL1A2 and COL2A1 genes are frequently reported in both non-lethal and lethal SD. The traditional prenatal diagnosis of SD using ultrasonography suffers from lower accuracy and performed at latter gestational stage. Therefore, it remains in desperate need of precise and accurate prenatal diagnosis of SD in early pregnancy. With the advancements of next-generation sequencing (NGS) technology and bioinformatics analysis, it is feasible to develop a NGS-based assay to detect genetic defects in association with SD in the early pregnancy.MethodsAn ampliseq-based targeted sequencing panel was designed to cover 87 recurrent hotspots reported in 11 common dominant SD and run on both Ion Proton and NextSeq550 instruments. Thirty-six cell-free and 23 genomic DNAs were used for assay developed. Spike-in DNA prepared from standard sample harboring known mutation and normal sample were also employed to validate the established SD workflow. Overall performances of coverage, uniformity, and on-target rate, and the detecting limitations on percentage of fetal fraction and read depth were evaluated.ResultsThe established targeted-seq workflow enables a single-tube multiplex PCR for library construction and shows high amplification efficiency and robust reproducibility on both Ion Proton and NextSeq550 platforms. The workflow reaches 100% coverage and both uniformity and on-target rate are > 96%, indicating a high quality assay. Using spike-in DNA with different percentage of known FGFR3 mutation (c.1138 G > A), the targeted-seq workflow demonstrated the ability to detect low-frequency variant of 2.5% accurately. Finally, we obtained 100% sensitivity and 100% specificity in detecting target mutations using established SD panel.ConclusionsAn expanded panel for rapid and cost-effective genetic detection of SD has been developed. The established targeted-seq workflow shows high accuracy to detect both germline and low-frequency variants. In addition, the workflow is flexible to be conducted in the majority of the NGS instruments and ready for routine clinical application. Taken together, we believe the established panel provides a promising diagnostic or therapeutic strategy for prenatal genetic testing of SD in routine clinical practice.

Abstract 2175: Identification of Somatic BRCA1/2 mutation profile in tissue-based and liquid biopsy-based next generation sequencing in Egyptian patients with breast cancer

Abstract Aim: Identification of BRCA1/2 mutations in Breast Cancer (BC) patients may help in providing genetic information to customize the therapeutic management of patients. Assessing liquid biopsies for BRCA1/2 somatic mutations could overcome the limitation of information gained from tissue biopsy. Consequently, this study aimed to identify the frequencies of pathogenic BRCA1 & BRCA2 somatic mutations in both liquid and tissue biopsies in Egyptian BC patients. Material and methods: The study was conducted on 45 BC patients. Liquid and tissue biopsies were collected from each patient and they were profiled for BRCA1/2 mutations using next generation sequencing (NGS). Qiaseq UMI-based targeted panel was used for performing libraries. Ion proton platform was used for sequencing. The detected genetic variants at depth of coverage 500x were annotated against Cosmic, dbSNP, and Exac all databases. Polyphen-2, Sift prediction tools and Clinvar database were used to detect the biological function of the detected variants using IVA software. Results: Variants analysis revealed that Among 45 patients, 37 (82.2%) patients were carrying one or more damaging BRCA1/2 somatic mutations. A total number of BRCA1/2 somatic variants detected was 24 and they were classified to 13 pathogenic, 7 likely pathogenic and 4 benign variants. Tissue and plasma samples of BC patients were found to harbor one detectable pathogenic somatic mutation in BRCA1 gene; c.1961delA with frequencies of 75.5% vs 17.7%, respectively. While, both samples had 8 detectable pathogenic BRCA2 somatic variants, the top 3 most frequent mutations among them were: c.5351delA (82.2% vs 40%), c.2957delA (75.56% vs 42.2%) and c.9253delA (68.89% vs 24.4%). One deleterious variant was detected merely in tissue sample; c.8940delA (55.5%). Whereas, 3 damaging variants were detected merely in plasma samples: c.8053delA (22.2%), c.6762delT (20%), c.4631delA (6.6%).Likely pathogenic BRCA2 somatic variants were also identified in both samples; c.7177delA (71.1% vs 53.3%) and c.10248delA (37.7% vs 11.1%). Moreover, 5 likely pathogenic variants were merely found in plasma samples. The most frequently detected variant among them was c.1114A>C (42.2%). Our data showed that exon 10 in BRCA1 and exon 11 in BRCA2 are the most affected exons as they harbored the most frequent detected pathogenic variants. Conclusion: Our preliminary results showed the most frequent pathogenic and likely pathogenic BRCA1/2 somatic mutations in liquid and tissue biopsies in Egyptian BC patients. This data may help in developing personalized targeted therapies that efficiently target the BRCA1/2 mutations. Citation Format: Mai M. Lotfy, Amira Salah El-Din Youssef, Auhood Nassar, Mahmoud Kamel, Zeinab K. Hassan, Amany Abd-Elhameed Abou-Bakr, Ahmed Farahat, Mohamed M. Gomaa, Abdel Rahman N. Zekri. Identification of Somatic BRCA1/2 mutation profile in tissue-based and liquid biopsy-based next generation sequencing in Egyptian patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2175.

The true incidence of chromosomal mosaicism after preimplantation genetic testing is much lower than that indicated by trophectoderm biopsy.

What is the true incidence of chromosomal mosaicism in embryos analyzed by preimplantation genetic testing (PGT). The true incidence of chromosomal mosaicism is much lower than we usually surmise. In recent years, contemporary methods for chromosome analysis, along with the biopsy of more than one cell, have given rise to an increased rate of chromosomal mosaicism detection after preimplantation genetic testing for aneuploidy. However, the exorbitant incidence of mosaicism represents a dilemma and imposes restrictions on the application of PGT treatment. Concern has been raised about the possibility that the incidence of chromosomal mosaicism is overestimated and quite a few of the results are false-positive errors. However, studies verifying the diagnosis of chromosomal mosaicism and assessing the true incidence of chromosomal mosaicism are limited. A total of 1719 blastocysts from 380 patients who underwent PGT treatment were retrospectively analyzed to evaluate the typical incidence of mosaicism. Then 101 embryos donated by 70 couples were re-biopsied and dissected into three portions if available: trophectoderm (TE), inner cell mass (ICM), and the remaining portions. All the portions were tested using next-generation sequencing (NGS), and the results were compared to the original diagnosis. The setting for this study was a university-affiliated center with an in-house PGT laboratory. All samples were amplified with multiple annealing and looping-based amplification cycles (MALBACs) and the NGS was carried out on a Life Technologies Ion Proton platform. A clinical TE biopsy revealed an incidence of 11.9% for diploid-aneuploid mosaicism (DAM), 17.3% for aneuploid mosaicism (AM) and 29.1% in total. After rebiopsy, 94.1% whole-chromosome aneuploidies and 82.8% segmental-chromosome aneuploidies were confirmed in the embryos. As for the mosaic errors, only 32 (31.7%) out of 101 embryos presented with uniform chromosomal aberrations in agreement with the original biopsy results, 15 (14.8%) embryos presented with de novo chromosomal aberrations, and 54 (53.5%) embryos showed a euploid profile in all portions. Among the 32 uniform embryos, the true mosaicism was confirmed in only 4 cases, where a reciprocal chromosomal aberration was identified; 14 embryos presented with identical mosaicism, providing the moderate evidence for true mosaicism; and 14 embryos displayed uniform full aneuploidies in all portions of embryo, revealing a high-grade mosaicism or a false-negative diagnosis. Logistical regression analysis revealed that the concordance rate with ICM was associated with the type and level of mosaicism. The concordance rate of segmental-chromosome mosaicism was significantly lower than whole-chromosome mosaicism (adjusted Odds Ratio (aOR): 5.137 (1.061, 24.876), P = 0.042) and compared to DAM, the concordance rate of AM was significantly higher (aOR: 6.546 (1.354, 31.655), P = 0.019). The concordance rate also increased with increasing levels of mosaicism (P < 0.001). This study was limited by a small sample size and the use of a single whole-genome amplification (WGA) method and NGS platform. These findings are only applicable to samples subjected to MALBAC amplification and Ion Proton platform, and studies involving larger sample sizes and multiple WGA methods and NGS platforms are required to prove our findings. TE biopsy is reliable to detect whole-chromosome aneuploidies, but the ability to diagnose mosaicism is doubtful. More attention should be paid to false-positive and false-negative errors in NGS-based PGT, especially for laboratories using less stringent criteria for mosaicism classification (i.e. 20-80%), which might be subject to a much higher false-positive mosaicism rate in the practice. This study was supported by grants from the National Key R&D Program of China (No. 2016YFC1000206-5) and the National Natural Science Foundation of China (No. 81701509). N/A.

Establishment and application of a method of next generation sequencing of 285 genes in lung cancer based on Ion-Proton platform.

BackgroundThe development of “precision medicine” needs a novel genetic screening and diagnostic technique for clinical detection. This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through testing 285 genes by customized next generation sequencing (NGS) on Ion-Proton platform.MethodsWe reviewed the related literature and collected data of genomic alteration that occurred in lung cancer. We identified 285 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. Targeted hybridization probes were designed using SureDesign software. The detection method was established by analyzing four cell lines and 13 lung cancer specimens which had been validated through Sanger sequencing. The sensitivity and specificity of the proposed method were preliminarily evaluated by comparisons with the Sanger sequencing and a LungCarta mutation-detection method.ResultsThe proposed method was able to detect mutations of 285 genes in lung cancer cell lines and clinical lung cancer specimens. The reads, mapped reads, on target, mean depth and uniformity were 14.90±4.37 (×106), 98.68%±0.61%, 60.49%±10.72%, 714.42±264.13 and 90.51%±6.91%, respectively. The detected mutation result of cell lines was consistent with the observations on previously reported mutations, and the congruence rate was 100%. The proposed method can detect single nucleotide polymorphism (SNP), InDel, Fusion and copy number variation (CNV). The complete congruence rate of detected result of specimens between the proposed method and Sanger sequencing, LungCarta mutation-detection method, immunohistochemistry (IHC), real-time polymerase chain reaction (RT-PCR) method were all 100% regarding mutations in common genes like EGFR, KRAS, or fusions of ALK, RET, etc. In addition, NFE2L3_p.Ser511_Pro513del, ERBB2_E770delinsEAYVM, MET_S701N, PDGFRA_T674I, TP53_G245V, TP53_V274A, TP53_A276F, TP53_G334L, TP53_R337L and TP53_Y220C mutations were detected only through the proposed method. The proposed method can detect mutations from blood, this detection result was consistent with the cancer tissues of the same clinical lung cancer patient.ConclusionsThe proposed Ion-Proton technology-based NGS method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method could be used to detect mutations in other cancer tissues and plasma, which needs further validation.

Abstract B091: Molecular alterations in black and Hispanic women with early breast cancer

Abstract Background: Black race is associated with inferior outcomes in early breast cancer, including hormone-receptor (HR) positive and HER2-negative disease, even when receiving comparable adjuvant systemic chemotherapy and endocrine therapy (Sparano et al. JNCI 2012; Albain et al. AACR SABCS 2018, abstract GS4-07). Hispanic ethnicity is associated with lower breast cancer incidence but higher mortality rates than non-Hispanic whites. Molecular differences in breast cancer could account for these disparities. A prior report based on Whole Exome Sequencing (WES) from the Cancer Genome Atlas (TCGA) in 105 African-American women showed higher rates of TP53 mutations and lower frequency of PIK3CA mutations (Keenan et al. JCO 2017), whereas data in Hispanics is limited. We use a custom sequencing panel called the Einstein Comprehensive Cancer Panel (ECCP) of ~150 genes commonly mutated in malignancies (Miller et al. Oncotarget 2017) in a cohort of 20 non-Hispanic black (NHB) and 24 Hispanic (H) women with early breast cancer. Methods: Ion AmpliSeq targeted libraries were prepared from genomic DNA isolated from breast tumor and adjacent non-tumor tissues and sequenced on the Ion Proton platform. Sequencing reads were analyzed using the Ion Torrent Suite’s Torrent Variant Caller followed by the Ion Reporter Software for variant calling and annotation. Results: Samples for 20 NHB and 24 H women were analyzed. The characteristics of the NHB/H cohorts were similar in age (median 55/59 years), stage (61%/73% with Stage II-III), and HR+/HER2-negative subtype (70%/77%). In NHB women, the most frequent pathogenic mutations were in TP53 (20%), PIK3CA (15%), BRCA1 (10%), CDH1 (10%), and KM2TC (10%). Of the NHB patients who recurred, 33% had a pathogenic mutation in TP53 and 16% in KRAS. Of NHB women with HR+ HER2- disease, the most common pathogenic mutations were in PIK3CA (21%) followed by CDH1, TP53, KM2TC, and BRCA1 (all 14%). In H women, the most frequent pathogenic mutations were in PIK3CA (50%) and TP53 (21%) followed by CDH1, FAT4, ATM, and KM2TC (all 8%). Of NHB women with HR+ HER2- disease, the most common pathogenic mutations were in PIK3CA (55%), TP53 (11%), and FAT4 (11%). Of the H patients who recurred, one had a deleterious mutation in TP53, BRCA1, and PIK3CA while the other had a deleterious mutation in GATA3. Conclusions: We found that the NHB and H patients had different molecular profiles despite similar clinical presentations with regard to age, stage, and subtype. The most notable finding was the very high rate of PIK3CA/PIK3R1 (50%) in breast cancers occurring in Hispanic women, which has implications for screening H women with metastatic breast cancer for PIK3CA-directed therapies such as alpelisib. The cohort has variants of unknown significance with mutations in genes that are not described in the literature as well as some hypermutated patients. This study indicates the importance of expanding molecular testing in diverse populations. Citation Format: Preethi Prasad, Cristina Montagna, Jesus D. Anampa Mesias, Maja Oktay, Joseph A. Sparano. Molecular alterations in black and Hispanic women with early breast cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B091.

Вплив ксенобіотиків на якісний склад прокаріот водної екосистеми Чорного моря

Процеси біотрансформації та біоремедіації, ключову роль у яких відіграють угруповання мікроорганізмів, виконують значну роль у вилученні токсичних речовин із трофічних ланцюгів екосистеми. Роль мікроорганізмів у розкладі ксенобіотиків є особливо важливою у водних екосистемах напівзакритого типу зі значним річковим стоком – таких, як Чорне море. Згідно з даними проєкту ПРООН ЕМБЛАС у водах Чорного моря було відмічено наявність низки поліароматичних вуглеводнів та хлорорганічних сполук, концентрація яких перевищувала гранично допустимі норми. Саме тому для дослідження функціональної відповіді екосистеми Чорного моря на вплив забруднюючих речовин, ми провели молекулярно-генетичну оцінку таксономічного біорізноманіття мікробних угруповань для виявлення таксонів, потенційно здатних до біотрансформації ксенобіотиків. Дослідні зразки морської води та донних осадів було відібрано в серпні-вересні 2017 року в рамках дослідницького рейсу (Joint Open Sea Survey) проєкту EMBLAS-II. Подальший аналіз включав екстракцію ДНК зі зразків та проведення секвенування на платформі Ion Proton длявизначеннятаксономічногобіорізноманіттямікробнихугруповань. Намибула показана наявність бактерій, що належать до таксономічних груп із попередньо експериментально визначеною здатністю до біотрансформації ксенобіотиків у водній товщі та донних осадах Чорного моря. Так, представники таких таксонів,як Rhodobacteraceae, Rhodospirillaceae, Halomonadaceae Oceanospirillaceae, Alcanivoracaceae, Bacilliaceae: Actinomycetales та Acidobacteriaceae були присутні в угрупованнях бактеріопланктону на всіх станціях дослідницького полігону. В угрупованнях донних осадів значну частку становили представники Alteromonadales та Piscirickettsiaceae, здатні до розкладу поліароматичних вуглеводнів, а також Desulfuromonadaceae, Desulfobacteraceae, Syntrophaceae, Peptococcaceae, Dehalococcoideaceae, для яких раніше була показана здатність до біотрансформації хлорорганічних сполук. Отримані результати є базовою інформацією, яка ляже в основу подальшого детального аналізу функціонального потенціалу мікробних угруповань щодо розкладу забруднюючих речовин, що має на меті ідентифікацію нових метаболічних шляхів біотрансформації ксенобіотиків та дослідження адаптаційних стратегій екосистеми до антропогенних змін навколишнього середовища.

Minority Gene Expression Profiling: Probing the Genetic Signatures of Pathogenesis Using Ribosome Profiling.

Minority Gene Expression Profiling (MGEP) refers to a scenario where the expression profiles of specific genes of interest are concentrated in a small cellular pool that is embedded within a larger, non-expressive pool. An example of this is the analysis of disease-related genes within sub-populations of blood or biopsied tissues. These systems are characterized by low signal-to-noise ratios that make it difficult, if not impossible, to uncover the desired signatures of pathogenesis in the absence of lengthy, and often problematic, technical manipulations. We have adapted ribosome profiling (RP) workflows from the Illumina to the Ion Proton platform and used them to analyze signatures of pathogenesis in an MGEP model system consisting of human cells eliciting <3% productive dengue infection. We find that RP is powerful enough to identify relevant responses of differentially expressed genes, even in the presence of significant noise. We discuss how to deal with sources of unwanted variation, and propose ways to further improve this powerful approach to the study of pathogenic signatures within MGEP systems.

Identifying miRNA Biomarkers and Predicted Targets Associated with Venous Thromboembolism in Colorectal Cancer Patients

Introduction Venous thromboembolism (VTE) is the second leading cause of mortality among cancer patients and is potentially preventable through the use of anticoagulation therapy. Risk models exist to guide use of prophylactic therapy but have low positive predictive value. New biomarkers are needed, particularly in intermediate-risk cancer types. Extracellular small RNAs, such as miRNAs, are promising biomarkers that have been implicated in tumor-dependent modification of platelets, a key component of thrombus formation. In this pilot study, we explore miRNAs as potential biomarkers for VTE risk in patients with colorectal cancer. Methods We conducted a case-control study utilizing specimens from a population enrolled in a prospective colorectal cancer biorepository at the Cleveland Clinic. Cases were defined as patients who developed VTE, including deep vein thrombosis and pulmonary embolism, within 6 months after cancer diagnosis and had their blood drawn prior to VTE. Cases were matched to controls (who did not have VTE and had a minimum of 6 months survival after cancer diagnosis) on a 1:2 ratio based on age, sex, cancer stage at diagnosis, and cancer treatment received prior to blood collection for a total of 21 patients. Total RNA from plasma specimens were sequenced on a Ion Proton platform (Thermo Fisher). Sequencing data were analyzed using the limma-voom R package. As this study was meant to be exploratory, miRNA were determined to be differentially expressed at a corrected Benjamini-Hochberg false detection rate (FDR) &amp;lt; 0.2. Target genes of differentially expressed miRNAs were predicted using mirDB and target gene pathways constructed with PANTHER. Results The study population had a median age of 65 (IQR 51-72). Of these patients, 85.7% were male; 42.9% had stage I/II cancer, 42.9% stage III cancer, 14.3% stage IV cancer; 71.4% had received no treatment prior to blood collection, 14.3% received chemo/chemoradiation therapy, and 14.3% received surgery. A total of 2426 unique miRNAs (median 1524, IQR 1371-1665) were expressed in the study group. Of these, 9 miRNAs were significantly differentially expressed (FDR &amp;lt; 0.2) and downregulated in cases compared to controls: hsa-miR-4451, 942-3p, 8063, 3132, 3118, 105-5p, 891a-5p, 200a-5p, and 6832-3p. From these miRNAs, 609 target genes were predicted and classified into 75 pathways, including angiogenesis, G-protein coupled receptors (GPCRs), inflammation mediated by chemo/cytokines, and integrin signaling. Target genes within these notable pathways included EPHA3, PDGFA, PTK2/FAK1, and IL15. Conclusions We identified 9 significantly downregulated miRNAs in the blood of colorectal cancer patients who developed VTE compared to controls in this pilot study. These data suggest that colorectal cancer patients may express unique miRNA profiles prior to VTE development which may be useful as biomarkers in future predictive models. In addition, this study identified potential new mechanistic targets for understanding cancer-associated thrombosis. While the role of GPCRs, integrins, and inflammation in platelet activation and function is widely known, this study also identified factors within the angiogenesis pathway that have been linked to increased platelet aggregation and tissue factor activation. Thus, downregulation of inhibitory miRNAs may cause disinhibition of pathways important for platelet and vascular function and other prothrombotic factors. Disclosures Khorana: Janssen: Consultancy; Bayer: Consultancy; Pfizer: Consultancy; Sanofi: Consultancy. McCrae:Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Whole exome sequencing identifies genomic alterations in proximal and distal colorectal cancer

Majority of colorectal cancer (CRC) patients are presented with advanced disease at diagnosis, particularly in cases of proximal CRCs. Little is known about the relationship between the genetic landscape and the anatomical location of the tumour; as well as the prognostication in CRC patients. The objectives of this study were to determine the somatic single nucleotide variants (SNV) and the cellular pathways between the proximal and distal CRCs. Whole exome sequencing was performed on the Ion Proton platform on 10 pairs of normal and CRC samples. The sequencing results were analysed using the Torrent Suite Software and the variants were annotated using ANNOVAR; followed by validation with Sanger sequencing. APC is the most frequently altered gene in both proximal and distal CRCs. KRAS and ATM genes were particularly altered in the proximal CRCs with a frequency of 60% and 40%, respectively. On the other hand, TP53 mutations did not show any CRC anatomical predominance. There were five recurrent novel variants in proximal CRCs and no recurrent variants identified in distal CRC. Wnt signalling pathway was the most frequently altered pathway in both proximal and distal CRCs whereas TGF-? and PI3K signalling pathways were predominantly altered in the proximal CRCs. We found that proximal CRCs presented with more variants and different altered pathways as compared to distal CRCs. However, further study in a larger series of samples coupled with functional studies will be required to confirm the identified variants and determine their roles in the pathogenesis of proximal and distal CRCs.

Saliva as a comparable-quality source of DNA for Whole Exome Sequencing on Ion platforms

BackgroundWhole Exome Sequencing (WES) utilises overlapping fragments prone to sequencing artefacts. Saliva, a non-invasive source of DNA, has been successfully used in WES studies on various platforms. This study explored the validity and quality of DNA sourced from saliva compared to whole blood on an Ion Platform. MethodsDNA was extracted from both sample types from four individuals. WES, performed on the Ion Proton platform was assessed for quality metrics (Depth, Genotyping Quality, etc.) and variant identification for the same source sample-pairs. ResultsNo significant differences in quality metrics were identified between data obtained from whole blood and saliva samples, with several saliva samples demonstrating higher coverage depth. Variants within the same sample, from the two genomic DNA sources, had an average concordance similar to other studies and platforms with different chemistry. ConclusionSaliva-extracted DNA provides comparable sequencing quality to whole blood for WES on Ion Torrent Platforms.

Specific clonal expansion at disease progression (PD) in solid cancers pinpointed by cell free DNA analysis.

e13144 Background: More than 50% of solid cancers sooner or later escape control of standard treatments. Detection and analysis of cell free circulating DNA (cfDNA) now offer the possibility to detect key mutations of cancer driver genes which may play a major role in the therapy escaping mechanism. We sought to identify clones of solid tumors escaping standard treatments in order to assess personalized treatment at PD. Methods: A cohort of patients with 10 different solid tumors progressing after standard therapy were selected. CfDNA analysis was performed using PAXgene blood ccfDNA tubes (QIAGEN), MagMAX cell-free total nucleic acid isolation kit, and ION PROTON platform (ThermoFisher Scientific). Results: Next generation sequencing analysis of 52 cancer-driver genes of cfDNA samples of 39 patients allowed for picking up clones plausibly involved in the PD mechanism in 60% of cases. A mean of 1.3 mutated genes (range 1-3) for each tumor was found. Point mutations in TP53, PIK3CA, and CNV in FGFR3 were the most commonly observed, with a rate of 41%, 16%, and 13%, respectively. Increased copy number variations of FGF receptors were identified in patients with non-small cell lung, pancreatic, and gastric cancer, and cholangiocarcinoma. Other clones had mutations in ESR1 (breast), CTNNB1 (uterus), KRAS and CCND2 (pancreas), EGFR and BRAF (lung). Interestingly, retinoblastomas resistant to Melphalan showed expanding mutated clones in PTEN or SMAD4. Increased levels of cfDNA were observed in the plasma of all patients. Conclusions: The results presented here show that irrespective of the primary tumor mutational burden and subsequent complex clonal evolution, a simplified mutational load is present at PD. One or few “sniper” clones drive progression and the molecular profile has a weak correlation with the primary tumor. Single driver mutations in TP53 remain the main target of a not yet developed specific therapy in most tumors such as breast, ovarian, uterine, lung, gastric cancers and glioblastoma. Among the actionable mutations, PIK3CA were found, not only in breast cancers, but also in uterine carcinoma, Sezary syndrome and glioblastoma, pinpointing the needs of specific trials in these tumors.

Whole exome sequencing identifies hemizygous deletions in the UGT2B28 and USP17L2 genes in a three‑generation family with endometriosis.

Endometriosis is an enigmatic condition with an unknown etiology and a poorly understood pathogenesis. It is considered to appear from the interplay of many genetic and environmental factors, affecting up to 10% of women and represents a major cause of pain and infertility. The familial association of endometriosis, as demonstrated through monozygotic twin and family studies suggests a genetic contribution to the disease, with further case-control and genome-wide association studies (GWAS) detecting various endometriosis risk factors. In a recent study, we described a unique, three-generation family of Cretan origin (Greece) with 7 females with surgically confirmed endometriosis (grandmother, 3 daughters and 3 granddaughters). All the affected members of this family displayed a variety of clinical manifestations and complications. In the present study, to further analyze the genetic variants conferring the risk of developing endometriosis, whole exome sequencing (WES) was performed, using the AmpliSeq technology on the Ion Proton platform. An initial analysis of 64 variants that were detected across the 14 genes previously confirmed to be associated with endometriosis, did not identify any deleterious exonic variants in these genes. However, further analysis revealed 2 hemizygous deletions in the grandmother that segregate in several of her affected offspring. The first deletion was found in the UGT2B28 locus, spanning 7 informative sequence variants across at least 14 kb. The second deletion, located in USP17L2, spans 3 informative variants across at least 2 kb. On the whole, the findings of the presents study implicate 2 additional genes in the pathogenesis of endometriosis, apart from those already identified by GWAS.

SU25 - TARGETED NEXT GENERATION SEQUENCING OF 187 GENES IN A BULGARIAN PSYCHOSIS SAMPLE

Background There is strong evidence for partial overlap of genetic influence on Schizophrenia (SCZ), Schizoaffective Disorder (SAD) and Bipolar Affective Disorder (BAD). Part of this is due to common genetic variants, detected by GWAS, with a small individual effect on risk. Recent studies using next generation sequencing reveal growing evidence of the role of rare genetic variants in both diseases. We performed targeted NGS on 187 candidate genes to further investigate the genetic architecture of Bulgarian patients with bipolar disorder and schizophrenia. Methods A total of 300 individuals with BAD, 151 with SCZ, 15 SAD according to DSMIV and ICD-10, as well as 85 healthy population controls and 40 healthy relatives were recruited. The samples were sequenced on the Ion PROTON platform. The sequencing panel comprised of 187 preselected strong candidate genes, based on the results from GWAS and previous NGS studies. Only samples with coverage of at least 95% of the target region at 20x were included in the analyses. Case-control association testing was done using PLINK. The rare variants have been evaluated and divided into groups based on their functional relevance: LOF variants (frameshift and nonsense); with potentially damaging effect on protein function (splice site, missense variants, 3’-UTR/5’-UTR variants); and with no known effect on function (synonymous and intron variants). Genes were prioritized by mutation burden analysis based on the average number of functional occurrences per 1000 bp of the gene CDS. The RVIS scoring system for assessing the intolerance of individual genes to protein-altering variants (Petrovski et al., 2013) was used in conjunction with the burden score for the final ordering. Results 5373 variants were detected, of which 2826 were found in affecteds (1831 singletons and 995 recurrent). No significant associations between cases and controls could be detected, that survived the correction for multiple testing. In total we found in patients 14 rare LOF variants; 889 non-synonymous variants (243 of which were potentially damaging); 109 splice site variants and 32 in regulatory regions. Discussion We could find no significant association of common or rare variants in the analyzed samples, most likely due to small sample size. However, we could identify both recurrent and singleton rare variants with potential functional relevance in genes associated with ion channels, postsynaptic plasticity, neurogenesis and signalling cascade pathways.

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

BackgroundSugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.MethodsWe generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.ResultsA total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.DiscussionThe new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

Analysis of Potential Molecular Targets inMonochamus alternatus(Coleoptera: Cerambycidae) Inoculated withBeauveria bassiana(Deuteromycotina: Hyphomycetes)

Abstract Monochamus alternatus Hope, the Japanese pine sawyer (Coleoptera: Cerambycidae), is a longhorn beetle that is a known vector of the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) Nickel. Beauveria bassiana (Balsamo) Vuillemin is an entomopathogenic fungus that is widely used as a microbial control agent because of its ease of mass production and safety to most vertebrates. To identify molecular targets that are potentially associated with B. bassiana toxicology, differentially expressed gene (DEG) libraries of M. alternatus contacted with B. bassiana have been prepared. The transcripts are sequenced using the Ion Proton platform; We identify 5,637, 9,181, and 1,787 sequences that involved cellular components, molecular functions, and biological processes, respectively. Fifty DEGs are enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, and 33 DEGs are enriched in insect hormone biosynthesis by a Kyoto Encyclopedia of Genes and Genomes pathway enrichment ana...

PO-482 Analysis of small RNA cargo in urinary and plasma EVs and matching prostate cancer and normal prostate tissues

IntroductionExtracellular vesicles (EVs) are membrane-enclosed structures released to the extracellular environment by different cell types, including cancer cells. Cancer derived-EVs have been shown to contain specific RNA cargo. Though, to what extent the EV cargo isolated from biofluids represents the tumour content is still under debate. The aim of this study was to characterise the small RNA content in plasma and urinary EVs collected from prostate cancer (PCa) patients before and after prostatectomy and to compare it with the matching tumour and normal tissue, in order to analyse their correlation and to identify RNA biomarkers that are released in plasma or urine from the tumour tissue.Material and methodsEVs were isolated from plasma and urine samples of 4 patients using size exclusion chromatography. EVs were quantified using Nanosight and their purity and size were assessed by electron microscopy and Western blot analysis. EVs were treated with Proteinase K and RNase A prior RNA isolation. Small RNA libraries were built and sequenced using Ion Proton and Illumina platforms. Specific miRNAs candidates were selected and validated by qPCR.Results and discussionsRNA deep sequencing analysis revealed that urinary EV RNA cargo is mainly comprised by miRNAs (51.68%), fragments of mRNAs (35.7%) and long non-coding RNAs (linc) (4.36%); while plasma EVs contained higher proportion of mRNA fragments (63.53%), miRNAs (12.31%) and processed pseudogenes (8.29%). Correlation analysis revealed low to moderate yet statistically significant correlation (≤0.4; p<0.001) between EV small RNA content and tumour tissue, while the correlation between EVs and normal prostate tissues was significantly lower. The highest correlation was observed between pre- and post-operation EV samples from the same biofluid (0.7–0.9; p<0.001). Among the different RNA species, miRNAs presented a moderate correlation with the tumour (≤0.3; p<0.001). A total of 822 different mature miRNAs were identified. A number of miRNAs overexpressed in tumour vs. adjacent normal tissue and present in urinary and plasma EVs were identified in each patient. From those, urinary derived miR-375 was validated and could serve as a biomarker for monitoring of PCa.ConclusionThis study provided a catalogue of small RNA species found in plasma and urinary EVs of PCa patients and revealed a number of miRNAs that are overexpressed in PCa and released in EVs.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Hippocampal Transcriptomic Profiles: Subfield Vulnerability to Age and Cognitive Impairment.

The current study employed next-generation RNA sequencing to examine gene expression differences related to brain aging, cognitive decline, and hippocampal subfields. Young and aged rats were trained on a spatial episodic memory task. Hippocampal regions CA1, CA3, and the dentate gyrus were isolated. Poly-A mRNA was examined using two different sequencing platforms, Illumina, and Ion Proton. The Illumina platform was used to generate seed lists of genes that were statistically differentially expressed across regions, ages, or in association with cognitive function. The gene lists were then retested using the data from the Ion Proton platform. The results indicate hippocampal subfield differences in gene expression and point to regional differences in vulnerability to aging. Aging was associated with increased expression of immune response-related genes, particularly in the dentate gyrus. For the memory task, impaired performance of aged animals was linked to the regulation of Ca2+ and synaptic function in region CA1. Finally, we provide a transcriptomic characterization of the three subfields regardless of age or cognitive status, highlighting and confirming a correspondence between cytoarchitectural boundaries and molecular profiling.

Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management

BackgroundNext generation sequencing technology has facilitated mapping of the colorectal cancer genotype and furthered our understanding of metastogenesis. The aim of this study was to investigate for conserved and different mutations in the exomes of synchronously resected primary colorectal tumour and liver metastases. This information could potentially be utilised to guide the treatment of advanced disease with the help of biological information from the primary tumour. MethodsWe performed exome sequencing of synchronously resected primary colorectal cancer and colorectal liver metastases as well as normal colonic mucosa and liver parenchyma, from four patients who had received neo-adjuvant chemotherapy, at a depth of 50X using the Ion Proton platform. Raw data was mapped to the reference genome prior to variant calling, annotation and downstream analysis. ResultsExome sequencing identified 585 non-synonymous missense single nucleotide variants (SNVs), of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. SNVs identified in the ErbB pathway appear to be concordant between primary and metastatic tumours. ConclusionOnly 13.8% of the metastatic exome can be predicted by the genotype of the primary tumour. We have demonstrated concordance of a number of SNVs in the ErbB pathway, which may inform selection of therapeutic agents in advanced colorectal cancer.