Search results for: nanopore sequencing

Authors: A. Delimitsou, C. Gouedard, E. Konstanta, A. Koletis, S. Patera, E. Manou, K. Spaho, S. Murray

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Background: There remains a lack of International Standard Control Reference materials for Next Generation Sequencing-based approaches or device calibration. We have designed and validated dsDNA biomimetic reference materials for targeted such approaches incorporating proprietary motifs (patent pending) for device/test calibration. They enable internal single-sample calibration, alleviating sample comparisons to pooled historical population-based data assembly or statistical modelling approaches. We have validated such an approach for BRCA Copy Number Variation analytics using iQRS™-CNVSUITE versus Mixed Ligation-dependent Probe Amplification. Methods: Standard BRCA Copy Number Variation analysis was compared between mixed ligation-dependent probe amplification and next generation sequencing using a cohort of 198 breast/ovarian cancer patients. Next generation sequencing based copy number variation analysis of samples spiked with iQRS™ dsDNA biomimetics were analysed using proprietary CNVSUITE software. Mixed ligation-dependent probe amplification analyses were performed on an ABI-3130 Sequencer and analysed with Coffalyser software. Results: Concordance of BRCA – copy number variation events for mixed ligation-dependent probe amplification and CNVSUITE indicated an overall sensitivity of 99.88% and specificity of 100% for iQRS™-CNVSUITE. The negative predictive value of iQRS-CNVSUITE™ for BRCA was 100%, allowing for accurate exclusion of any event. The positive predictive value was 99.88%, with no discrepancy between mixed ligation-dependent probe amplification and iQRS™-CNVSUITE. For device calibration purposes, precision was 100%, spiking of patient DNA demonstrated linearity to 1% (±2.5%) and range from 100 copies. Traditional training was supplemented by predefining the calibrator to sample cut-off (lock-down) for amplicon gain or loss based upon a relative ratio threshold, following training of iQRS™-CNVSUITE using spiked iQRS™ calibrator and control mocks. BRCA copy number variation analysis using iQRS™-CNVSUITE™ was successfully validated and ISO15189 accredited and now enters CE-IVD performance evaluation. Conclusions: The inclusion of a reference control competitor (iQRS™ dsDNA mimetic) to next generation sequencing-based sequencing offers a more robust sample-independent approach for the assessment of copy number variation events compared to mixed ligation-dependent probe amplification. The approach simplifies data analyses, improves independent sample data analyses, and allows for direct comparison to an internal reference control for sample-specific quantification. Our iQRS™ biomimetic reference materials allow for single sample copy number variation analytics and further decentralisation of diagnostics to single patient sample assessment.

Keywords: validation, diagnostics, oncology, copy number variation, reference material, calibration

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Authors: Maryam Vaezjalali, Koroush Rahimian, Maryam Asli, Tahmineh Kandelouei, Foad Davoodbeglou, Amir H. Kashi

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Objectives: The present study was conducted to assess the HBV molecular profiles including genotypes, subgenotypes, subtypes & mutations in hepatitis B genes. Materials/Patients and Methods: This study was conducted on 229 intravenous drug users who referred to three Drop- in-Centers and a hospital in Tehran. HBV DNA was extracted from HBsAg positive serum samples and amplified by Nested PCR. HBV genotype, subgenotypes, subtype and genes mutation were determined by direct sequencing. Phylogenetic tree was constructed using neighbor- joining (NJ) method. Statistical analyses were carried out by SPSS 20. Results: HBV DNA was found in 3 HBsAg positive cases. Phylogenetic tree of derived HBV DNAs showed the existence of genotype D (subgenotype D1, subtype ayw2). Also immune escape mutations were determined in S gene. Conclusion: There were a few variations and genotypes and subtypes among infected intravenous drug users. This study showed the predominance of genotype D among intravenous drug users. Our study concurs with other reports from Iran, that all showing currently only genotype D is the only detectable genotype in Iran.

Keywords: drug users, genotype, HBV, phylogenetic tree

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Authors: Tanushree Jaitly, Shailendra Gupta, Leila Taher, Gerold Schuler, Julio Vera

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Genomics-based personalized medicine is a promising approach to fight aggressive tumors based on patient's specific tumor mutation and expression profiles. A remarkable case is, dendritic cell-based immunotherapy, in which tumor epitopes targeting patient's specific mutations are used to design a vaccine that helps in stimulating cytotoxic T cell mediated anticancer immunity. Here we present a computational pipeline for epitope-based personalized cancer vaccines using patient-specific haplotype and cancer mutation profiles. In the workflow proposed, we analyze Whole Exome Sequencing and RNA Sequencing patient data to detect patient-specific mutations and their expression level. Epitopes including the tumor mutations are computationally predicted using patient's haplotype and filtered based on their expression level, binding affinity, and immunogenicity. We calculate binding energy for each filtered major histocompatibility complex (MHC)-peptide complex using docking studies, and use this feature to select good epitope candidates further.

Keywords: cancer immunotherapy, epitope prediction, NGS data, personalized medicine

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Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

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Authors: Young Ah Kim, Hyunsoo Kim, Eun-Jeong Yoon, Young Hee Seo, Kyungwon Lee

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Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from food animals are considered as a reservoir for transmission of ESBL genes to human. The aim of this study is to assess the prevalence and molecular epidemiology of ESBL-producing E. coli colonization in pigs, farm workers, and farm environments to elucidate the transmission of multidrug-resistant clones from animal to human. Nineteen pig farms were enrolled across the country in Korea from August to December 2017. ESBL-producing E. coli isolates were detected in 190 pigs, 38 farm workers, and 112 sites of farm environments using ChromID ESBL (bioMerieux, Marcy l'Etoile, France), directly (stool or perirectal swab) or after enrichment (sewage). Antimicrobial susceptibility tests were done with disk diffusion methods and blaTEM, blaSHV, and blaCTX-M were detected with PCR and sequencing. The genomes of the four CTX-M-55-producing E. coli isolates from various sources in one farm were entirely sequenced to assess the relatedness of the strains. Whole genome sequencing (WGS) was performed with PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). ESBL genotypes were 85 CTX-M-1 group (one CTX-M-3, 23 CTX-M-15, one CTX-M-28, 59 CTX-M-55, one CTX-M-69) and 60 CTX-M-9 group (41 CTX-M-14, one CTX-M-17, one CTX-M-27, 13 CTX-M-65, 4 CTX-M-102) in total 145 isolates. The rectal colonization rates were 53.2% (101/190) in pigs and 39.5% (15/38) in farm workers. In WGS, sequence types (STs) were determined as ST69 (E. coli PJFH115 isolate from a human carrier), ST457 (two E. coli isolates PJFE101 recovered from a fence and PJFA1104 from a pig) and ST5899 (E. coli PJFA173 isolate from the other pig). The four plasmids encoding CTX-M-55 (88,456 to 149, 674 base pair), whether it belonged to IncFIB or IncFIC-IncFIB type, shared IncF backbone furnishing the conjugal elements, suggesting of genes originated from same ancestor. In conclusion, the prevalence of ESBL-producing E. coli in swine farms was surprisingly high, and many of them shared common ESBL genotypes of clinical isolates such as CTX-M-14, 15, and 55 in Korea. It could spread by horizontal transfer between isolates from different reservoirs (human-animal-environment).

Keywords: Escherichia coli, extended-spectrum β-lactamase, prevalence, whole genome sequencing

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Authors: Barsha Saha, Shouvik Chakravarty, Sukanta Ray, Kshaunish Das, Nidhan K. Biswas, Srikanta Goswami

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Pancreatic Ductal Adenocarcinoma is a well-recognised cause of cancer death with a five-year survival rate of about 9%, and its incidence in India has been found to be increased manifold in recent years. Due to delayed detection, this highly metastatic disease has a poor prognosis. Several molecular alterations happen during the progression of the disease from pre-cancerous conditions, and many such alterations could be investigated for their biomarker potential. MicroRNAs have been shown to be prognostic for PDAC patients in a variety of studies. We hereby used NGS technologies to evaluate the role of small RNA changes during pancreatic cancer development from chronic pancreatitis. Plasma samples were collected from pancreatic cancer patients (n=16), chronic pancreatitis patients (n=8), and also from normal individuals (n=16). Pancreatic tumour tissue (n=5) and adjacent normal tissue samples (n=5) were also collected. Sequencing of small RNAs was carried out after small RNAs were isolated from plasma samples and tissue samples. We find that certain microRNAs are highly deregulated in pancreatic cancer patients in comparison to normal samples. A combinatorial analysis of plasma and tissue microRNAs and subsequent exploration of their targets and altered molecular pathways could not only identify potential biomarkers for disease diagnosis but also help to understand the underlying mechanism.

Keywords: small RNA sequencing, pancreatic cancer, biomarkers, tissue sample

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Authors: Yantao Li, Jun Zheng

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Food poison caused by consumption of contaminated food, especially seafood, is one of most serious public health threats worldwide. Vibrio parahaemolyticus is emerging bacterial pathogen and the leading cause of human gastroenteritis associated with food poison, especially in the southern coastal region of China. To successfully cause disease in host, bacterial pathogens need to overcome the host-derived stresses encountered during infection. One of the toxic chemical species elaborated by the host is nitric oxide (NO). NO is generated by acidified nitrite in the stomach and by enzymes of the inducible NO synthase (iNOS) in the host cell, and is toxic to bacteria. Bacterial pathogens have evolved some mechanisms to battle with this toxic stress. Such mechanisms include genes to sense NO produced from immune system and activate others to detoxify NO toxicity, and genes to repair the damage caused by toxic reactive nitrogen species (RNS) generated during NO toxic stress. However, little is known about the NO resistance in V. parahaemolyticus. In this study, a transposon coupled with next generation sequencing (Tn-seq) technology will be utilized to identify genes for NO resistance in V. parahaemolyticus. Our strategy will include construction the saturating transposon insertion library, transposon library challenging with NO, next generation sequencing (NGS), bioinformatics analysis and verification of the identified genes in vitro and in vivo.

Keywords: vibrio parahaemolyticus, nitric oxide, tn-seq, virulence

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Authors: Sijie Chen, Xiaofang Chen, Juan Wang, Yingying Zhang, Yu Hong, Wanyu Zhuang, Xinxin Huang, Ping Ou, Longsheng Huang

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Purpose: Autistic symptom improvement can be observed in children treated with acupuncture, but the mechanism is still being explored. In the present study, we used scalp acupuncture to treat autism rat model, and then their improvement in the abnormal behaviors and specific mechanisms behind were revealed by detecting animal behaviors, analyzing the RNA sequencing of the prefrontal cortex(PFC), and observing the ultrastructure of PFC neurons under the transmission electron microscope. Methods: On gestational day 12.5, Wistar rats were given valproic acid (VPA) by intraperitoneal injection, and their offspring were considered to be reliable rat models of autism. They were randomized to VPA or VPA-acupuncture group (n=8). Offspring of Wistar pregnant rats that were simultaneously injected with saline were randomly selected as the wild-type group (WT). VPA_acupuncture group rats received acupuncture intervention at 23 days of age for 4 weeks, and the other two groups followed without intervention. After the intervention, all experimental rats underwent behavioral tests. Immediately afterward, they were euthanized by cervical dislocation, and their prefrontal cortex was isolated for RNA sequencing and transmission electron microscopy. Results: The main results are as follows: 1. Animal behavioural tests: VPA group rats showed more anxiety-like behaviour and repetitive, stereotyped behaviour than WT group rats. While VPA group rats showed less spatial exploration ability, activity level, social interaction, and social novelty preference than WT group rats. It was gratifying to observe that acupuncture indeed improved these abnormal behaviors of autism rat model. 2. RNA-sequencing: The three groups of rats differed in the expression and enrichment pathways of multiple genes related to synaptic function, neural signal transduction, and circadian rhythm regulation. Our experiments indicated that acupuncture can alleviate the major symptoms of ASD by improving these neurological abnormalities. 3. Under the transmission electron microscopy, several lysosomes and mitochondrial structural abnormalities were observed in the prefrontal neurons of VPA group rats, which were manifested as atrophy of the mitochondrial membran, blurring or disappearance of the mitochondrial cristae, and even vacuolization. Moreover, the number of synapses and synaptic vesicles was relatively small. Conversely, the mitochondrial structure of rats in the WT group and VPA_acupuncture was normal, and the number of synapses and synaptic vesicles was relatively large. Conclusion: Acupuncture effectively improved the abnormal behaviors of autism rat model and the ultrastructure of the PFC neurons, which might worked by improving their abnormal synaptic function, synaptic plasticity and promoting neuronal signal transduction.

Keywords: autism spectrum disorder, acupuncture, animal behavior, RNA sequencing, transmission electron microscope

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Authors: Didem Güven, Oytun Hanhan, Elif Ceren Aksoy, Emine Ubay Çokgör

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This paper presents a biological treatability study ofpaintshopwastewaterof a bus factory by an anoxic/aerobic sequencing batch reactor.A lab scale 14L SBR system was implementedto investigate carbon and nitrogen removal performance frompaint shop waste streams combined with domestic and process wastewater of a bus production factory in Istanbul (Turkey).The wastewater collected from decanters of the paint boots and pre-treatmentplant was usedforthefeeding of SBR. The reactor was operated with a total hydraulic retention time of 24 hrs, and a total sludge age of 18.7 days. Initially the efficiency and stability of the reactor were studied when fed with main wastewater stream to simulate the current wastewater treatment plant. Removal efficiency of 57% nitrogen and 90% COD were obtained. Once the paint shop wastewater was introduced to mainstream feeding with a ratio of 1:5, nitrification completely, carbon removal were partially inhibited. SBR system was successful to handle even at very high COD concentrations of paint shop wastewater after feeding of 2 months, with an average effluent COD of 100 mg/L. For the determination of kinetic parameters, respirometric analysis was also conducted with/without paint shop wastewater addition. Model simulation indicated lower maximum specific growth and hydrolysis rates when paint shop wastewater was mixed with the mainstream wastewater of the factory.

Keywords: biological treatability, nitrogen removal, paint shop wastewater, sequencing batch reactor

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Authors: Rozhgar A. Khailany, Mehri Igci, Emine Bayraktar, Sakip Erturhan, Metin Karakok, Ahmet Arslan

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Kidney cancer is the most lethal urological cancer accounting for 3% of adult malignancies. VHL, a tumor-suppressor gene, is best known to be associated with renal cell carcinoma (RCC). The VHL functions as negative regulator of hypoxia inducible factors. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC (clear cell RCC) including PBRM1 and SETD2. The PBRM1 gene encodes the BAF180 protein, which involved in transcriptional activation and repression of selected genes. SETD2 encodes a histone methyltransferase, which may play a role in suppressing tumor development. In this study, RNAs of 30 paired tumor and normal samples that were grouped according to the types of kidney cancer and clinical characteristics of patients, including gender and average age were examined by RT-PCR, SSCP and sequencing techniques. VHL, PBRM1 and SETD2 expressions were relatively down-regulated. However, statistically no significance was found (Wilcoxon signed rank test, p > 0.05). Interestingly, no mutation was observed on the contrary of previous studies. Understanding the molecular mechanisms involved in the pathogenesis of RCC has aided the development of molecular-targeted drugs for kidney cancer. Further analysis is required to identify the responsible genes rather than VHL, PBRM1 and SETD2 in kidney cancer.

Keywords: kidney cancer, molecular biomarker, expression analysis, mutation screening

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Authors: M. Suwaibah, S. W. Tan, I. Aiini, K. Yusoff, A. R. Omar

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Respiratory diseases are the most important infectious diseases affecting poultry worldwide. One of the avian respiratory virus of global importance causing significant economic losses is Infectious Bronchitis Virus (IBV). The virus causes a wide spectrum disease known as Infectious Bronchitis (IB), affecting not only the respiratory system but also the kidney and the reproductive system, depending on its strain. IB and Newcastle disease are two of the most prevalent diseases affecting poultry in Malaysia. However, a study on the molecular characterization of Malaysian IBV is lacking. In this study, an IBV strain IBS130 which was isolated in 2015 was fully sequenced using next-gene sequencing approach. Sequence analysis of IBS130 based on the complete genome, polyprotein 1ab and S1 genes were compared with other IBV sequences available in Genbank, National Center for Biotechnology Information (NCBI). IBV strain IBS130 is characterised as QX-like strain based on whole genome and S1 gene sequence analysis. Comparisons of the virus with other IBV strains showed that the nucleotide identity ranged from 67% to 99.2%, depending on the region analysed. The similarity in whole genome nucleotide ranging from 84.9% to 90.7% with the least similar was from Singapore strains (84.9%) and highly similar with China QX-like strains. Meanwhile, the similarity in polyprotein 1ab ranging from 85.3% to 89.9% with the least similar to Singapore strains (85.3%) and highly similar with Mass strains from USA.

Keywords: infectious bronchitis virus, phylogenetic analysis, chicken, Malaysia

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Authors: Adriana Sosa, Susana Rincon, Chérif Ben, Diana Cabañas, Juan E. Ruiz, Alejandro Zepeda

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The complex chemical composition (mixtures of ammonium and recalcitrant compounds) of the effluents from the chemical, pharmaceutical and petrochemical industries represents a challenge in their biological treatment. This treatment involves nitrification process that can suffer an inhibition due to the presence of aromatic compounds giving as a result the decrease of the process efficiency. The inhibitory effects on nitrification in the presence of aromatic compounds have already been studied; however a few studies have considered the presence of phenolic compounds in the form of mixtures, which is the form that they are present in real context. For this reason, we realized a kinetic study on the nitrifying process in the presence of different concentrations of a mixture of phenol, o-cresol, m-cresol and p-cresol (0 - 320 mg C/L) in a sequencing batch reactor (SBR). Firstly, the nitrifying process was evaluated in absence of the phenolic mixture (control 1) in a SBR with 2 L working volume and 176 mg/L of nitrogen of microbial protein. Total oxidation of initial ammonium (efficiency; ENH4+ of 100 %) to nitrate (nitrifying yield; YNO3- of 0.95) were obtained with specific rates of ammonium consumption (qN-NH4+) and nitrate production (qN-NO3-) (of 1.11 ± 0.04 h-1 and 0.67 h-1 ± 0.11 respectively. During the phase of acclimation with 40 mg C/L of the phenolic mixture, an inhibitory effect on the nitrifying process was observed, provoking a decrease in ENH4+ and YNO3- (11 and 54 % respectively) as well as in the specific rates (89 y 46 % respectively), being the ammonia oxidizing bacteria (BAO) the most affected. However, in the next cycles without the phenolic mixture (control 2), the nitrifying consortium was able to recover its nitrifying capacity (ENH4+ = 100% and YNO3-=0.98). Afterwards the SBR was fed with 10 mg C/L of the phenolic mixture, obtaining and ENH4+ of 100%, YNO3- and qN-NH4+ 0.62 ± 0.006 and 0.13 ± 0.004 respectively, while the qN-NO3- was 0.49 ± 0.007. Moreover, with the increase of the phenolic concentrations (10-160 mg C/L) and the number of cycles the nitrifying consortium was able to oxidize the ammonia with ENH4+ of 100 % and YNO3- close to 1. However a decrease in the values of the nitrification specific rates and increase in the oxidation in phenolic compounds (70 to 94%) were observed. Finally, in the presence of 320 mg C/L, the nitrifying consortium was able to simultaneously oxidize the ammonia (ENH4+= 100%) and the phenolic mixture (p-cresol>phenol>m-cresol>o-cresol) being the o-cresol the most recalcitrant compound. In all the experiments the use of a SBR allowed a respiratory adaptation of the consortium to oxidize the phenolic mixture achieving greater adaptation of the nitrite-oxidizing bacteria (NOB) than in the ammonia-oxidizing bacteria (AOB).

Keywords: cresol, inhibition, nitrification, phenol, sequencing batch reactor

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Authors: Omar Youssef, Aija Knuuttila, Paivi Piirilä, Virinder Sarhadi, Sakari Knuutila

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Exhaled breath condensate (EBC) is a unique sample that allows studying different genetic changes in lung carcinoma through a non-invasive way. With the aid of next generation sequencing (NGS) technology, analysis of genetic mutations has been more efficient with increased sensitivity for detection of genetic variants. In order to investigate the possibility of applying this method for cancer diagnostics, mutations in EBC DNA from lung cancer patients and healthy individuals were studied by using NGS. The key aim is to assess the feasibility of using this approach to detect clinically important mutations in EBC. EBC was collected from 20 healthy individuals and 9 lung cancer patients (four lung adenocarcinomas, four 8 squamous cell carcinoma, and one case of mesothelioma). Mutations in hotpot regions of 22 genes were studied by using Ampliseq Colon and Lung cancer panel and sequenced on Ion PGM. Results demonstrated that all nine patients showed a total of 19 cosmic mutations in APC, BRAF, EGFR, ERBB4, FBXW7, FGFR1, KRAS, MAP2K1, NRAS, PIK3CA, PTEN, RET, SMAD4, and TP53. In controls, 15 individuals showed 35 cosmic mutations in BRAF, CTNNB1, DDR2, EGFR, ERBB2, FBXW7, FGFR3, KRAS, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, and TP53. Additionally, 45 novel mutations not reported previously were also seen in patients’ samples, and 106 novel mutations were seen in controls’ specimens. KRAS exon 2 mutations G12D was identified in one control specimen with mutant allele fraction of 6.8%, while KRAS G13D mutation seen in one patient sample showed mutant allele fraction of 17%. These findings illustrate that hotspot mutations are present in DNA from EBC of both cancer patients and healthy controls. As some of the cosmic mutations were seen in controls too, no firm conclusion can be drawn on the clinical importance of cosmic mutations in patients. Mutations reported in controls could represent early neoplastic changes or normal homeostatic process of apoptosis occurring in lung tissue to get rid of mutant cells. At the same time, mutations detected in patients might represent a non-invasive easily accessible way for early cancer detection. Follow up of individuals with important cancer mutations is necessary to clarify the significance of these mutations in both healthy individuals and cancer patients.

Keywords: exhaled breath condensate, lung cancer, mutations, next generation sequencing

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Authors: Amr Saeb, Khalid Al-Rubeaan, Mohamed Abouelhoda, Manojkumar Selvaraju, Hamsa Tayeb

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Background: P. mirabilis is a common uropathogenic bacterium that can cause major complications in patients with long-standing indwelling catheters or patients with urinary tract anomalies. In addition, P. mirabilis is a common cause of chronic osteomyelitis in diabetic foot ulcer (DFU) patients. Methodology: P. mirabilis SCDR1 was isolated from a diabetic ulcer patient. We examined P. mirabilis SCDR1 levels of resistance against nano-silver colloids, the commercial nano-silver and silver containing bandages and commonly used antibiotics. We utilized next generation sequencing techniques (NGS), bioinformatics, phylogenetic analysis and pathogenomics in the identification and characterization of the infectious pathogen. Results: P. mirabilis SCDR1 is a multi-drug resistant isolate that also showed high levels of resistance against nano-silver colloids, nano-silver chitosan composite and the commercially available nano-silver and silver bandages. The P. mirabilis-SCDR1 genome size is 3,815,621 bp with G+C content of 38.44%. P. mirabilis-SCDR1 genome contains a total of 3,533 genes, 3,414 coding DNA sequence genes, 11, 10, 18 rRNAs (5S, 16S, and 23S), and 76 tRNAs. Our isolate contains all the required pathogenicity and virulence factors to establish a successful infection. P. mirabilis SCDR1 isolate is a potential virulent pathogen that despite its original isolation site, wound, it can establish kidney infection and its associated complications. P. mirabilis SCDR1 contains several mechanisms for antibiotics and metals resistance including, biofilm formation, swarming mobility, efflux systems, and enzymatic detoxification. Conclusion: P. mirabilis SCDR1 is the spontaneous nano-silver resistant bacterial strain. P. mirabilis SCDR1 strain contains all reported pathogenic and virulence factors characteristic for the species. In addition, it possesses several mechanisms that may lead to the observed nano-silver resistance.

Keywords: Proteus mirabilis, multi-drug resistance, silver nanoparticles, resistance, next generation sequencing techniques, genome analysis, bioinformatics, phylogeny, pathogenomics, diabetic foot ulcer, xenobiotics, multidrug resistance efflux, biofilm formation, swarming mobility, resistome, glutathione S-transferase, copper/silver efflux system, altruism

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Authors: Arati Inamdar, Siddharth Bhattacharyya, Kester Haye

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Collision tumors are rare entities characterized by neoplasms of two different cell populations with distinct separating boundaries. Such tumors could be benign, malignant, or a combination of both. The exact mechanism of origin for collision tumors is predicted to be tumor heterogeneity or concurrent occurrence of neoplasm in the same organ. We present two cases of plasmacytoma presenting as a collision tumor, one with a tumor of hematological origin and another with a non-hematological origin, namely Chronic Lymphocytic Leukemia and Adenocarcinoma of the colon, respectively. The immunohistochemical stains and flowcytometry analysis performed on the specimens aided incorrect diagnosis. Interestingly, neoplastic cells of plasmacytoma in the first case demonstrated strong cytokeratin along with weak Epithelial Specific Antigen/ Epithelial cell adhesion molecule Monoclonal Antibody (MOC31) positivity, indicating that the tumor may influence the microenvironment of the tumor in the vicinity. Furthermore, the next-generation sequencing studies performed on the specimen with plasmacytoma and chronic lymphocytic lymphoma demonstrated BReast CAncer gene (BRCA2) and Tumor Necrosis Factor Alpha Induced Protein 3 (TNFAIP3) as a disease associated variants suggestive of risk for multiple tumors including collision tumors. Our reports highlight the unique collision tumors involving plasmacytoma, which have never been reported previously, as well as provide necessary insights about the underline genetic aberrations and tumor heterogeneity through sequencing studies and allow clonality assessment for subsequent tumors.

Keywords: BRCA2, collision tumor, chronic lymphocytic leukemia, plasmacytoma

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Authors: Muhammad Sohail Sajid, Qurat-ul-Ain, Zafar Iqbal, Muhammad Nisar Khan, Asma Kausar, Adil Ejaz

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Rickettsiosis, caused by a Spotted Fever Group Rickettsiae (SFGR), is considered as an emerging infectious disease from public and veterinary perspective. The present study reports distribution of SFGR in the host (buffalo and cattle) and vector (ticks) population determined through gene specific amplification through PCR targeting outer membrane protein (ompA). Tick and blood samples were collected using standard protocols through convenient sampling from district Faisalabad. Ticks were dissected to extract salivary glands (SG). Blood and tick SG pools were subjected to DNA extraction and amplification of ompA using PCR. Overall prevalence of SFGR was reported as 21.5% and 33.6 % from blood and ticks, respectively. Hyalomma anatolicum was more prevalent tick associated with SFGR as compared to Rhipicephalus sp. Higher prevalence of SFGR was reported in cattle (25%) population as compared to that of buffalo (17.07%). On seasonal basis, high SFGR prevalence was recorded during spring season (48.1%, 26.32%, 17.76%) as compared to winter (27.9%, 21.43%, 15.38%) in vector and host (cattle and buffalo respectively) population. Sequencing analysis indicated that rickettsial endo-symbionts were associated with ticks of the study area. These results provided baseline information about the prevalence of SFGR in vector and host population.

Keywords: Rickettsia, livestock, polymerase chain reaction, sequencing, ticks, vectors

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Authors: Leo Nnamdi Ozurumba-Dwight

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Single-cell genomic analytical systems have proved to be a platform to isolate bulk cells into selected single cells for genomic, proteomic, and related metabolomic studies. This is enabling systematic investigations of the level of heterogeneity in a diverse and wide pool of cell populations. Single cell technologies, embracing techniques such as high parameter flow cytometry, single-cell sequencing, and high-resolution images are playing vital roles in these investigations on messenger ribonucleic acid (mRNA) molecules and related gene expressions in tracking the nature and course of disease conditions. This entails targeted molecular investigations on unit cells that help us understand cell behavoiur and expressions, which can be examined for their health implications on the health state of patients. One of the vital good sides of single-cell RNA sequencing (scRNA seq) is its probing capacity to detect deranged or abnormal cell populations present within homogenously perceived pooled cells, which would have evaded cursory screening on the pooled cell populations of biological samples obtained as part of diagnostic procedures. Despite conduction of just single-cell transcriptome analysis, scRNAseq now permits comparison of the transcriptome of the individual cells, which can be evaluated for gene expressional patterns that depict areas of heterogeneity with pharmaceutical drug discovery and clinical treatment applications. It is vital to strictly work through the tools of investigations from wet lab to bioinformatics and computational tooled analyses. In the precise steps for scRNAseq, it is critical to do thorough and effective isolation of viable single cells from the tissues of interest using dependable techniques (such as FACS) before proceeding to lysis, as this enhances the appropriate picking of quality mRNA molecules for subsequent sequencing (such as by the use of Polymerase Chain Reaction machine). Interestingly, scRNAseq can be deployed to analyze various types of biological samples such as embryos, nervous systems, tumour cells, stem cells, lymphocytes, and haematopoietic cells. In haematopoietic cells, it can be used to stratify acute myeloid leukemia patterns in patients, sorting them out into cohorts that enable re-modeling of treatment regimens based on stratified presentations. In immunotherapy, it can furnish specialist clinician-immunologist with tools to re-model treatment for each patient, an attribute of precision medicine. Finally, the good predictive attribute of scRNAseq can help reduce the cost of treatment for patients, thus attracting more patients who would have otherwise been discouraged from seeking quality clinical consultation help due to perceived high cost. This is a positive paradigm shift for patients’ attitudes primed towards seeking treatment.

Keywords: immunotherapy, transcriptome, re-modeling, mRNA, scRNA-seq

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Authors: Maxence Queyrel, Edi Prifti, Alexandre Templier, Jean-Daniel Zucker

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Analysis of the human microbiome using metagenomic sequencing data has demonstrated high ability in discriminating various human diseases. Raw metagenomic sequencing data require multiple complex and computationally heavy bioinformatics steps prior to data analysis. Such data contain millions of short sequences read from the fragmented DNA sequences and stored as fastq files. Conventional processing pipelines consist in multiple steps including quality control, filtering, alignment of sequences against genomic catalogs (genes, species, taxonomic levels, functional pathways, etc.). These pipelines are complex to use, time consuming and rely on a large number of parameters that often provide variability and impact the estimation of the microbiome elements. Training Deep Neural Networks directly from raw sequencing data is a promising approach to bypass some of the challenges associated with mainstream bioinformatics pipelines. Most of these methods use the concept of word and sentence embeddings that create a meaningful and numerical representation of DNA sequences, while extracting features and reducing the dimensionality of the data. In this paper we present an end-to-end approach that classifies patients into disease groups directly from raw metagenomic reads: metagenome2vec. This approach is composed of four steps (i) generating a vocabulary of k-mers and learning their numerical embeddings; (ii) learning DNA sequence (read) embeddings; (iii) identifying the genome from which the sequence is most likely to come and (iv) training a multiple instance learning classifier which predicts the phenotype based on the vector representation of the raw data. An attention mechanism is applied in the network so that the model can be interpreted, assigning a weight to the influence of the prediction for each genome. Using two public real-life data-sets as well a simulated one, we demonstrated that this original approach reaches high performance, comparable with the state-of-the-art methods applied directly on processed data though mainstream bioinformatics workflows. These results are encouraging for this proof of concept work. We believe that with further dedication, the DNN models have the potential to surpass mainstream bioinformatics workflows in disease classification tasks.

Keywords: deep learning, disease prediction, end-to-end machine learning, metagenomics, multiple instance learning, precision medicine

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Authors: Pasquali Frederique, Manfreda Chiara, Crippa Cecilia, Indio Valentina, Ianieri Adriana, De Cesare Alessandra

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Microbiological hazards can be transmitted to humans through milk. In this study, we compared the microbiome composition and presence of foodborne pathogens in organic milk (n=6), organic hay milk (n=6), standard milk (n=6) and high-quality milk (n=6). The milk samples were collected during six samplings between December 2022 to January 2023 and between April and May 2024 to take into account seasonal variations. The 24 milk samples were submitted to DNA extraction and library preparation before shotgun sequencing on the Illumina HiScan™ SQ System platform. The total sequencing output was 600 GB. In all the milk samples, the phyla with the highest relative abundances were Pseudomonadota, Bacillota, Ascomycota, Actinomycetota and Apicomplexa, while the most represented genera were Pseudomonas, Streptococcus, Geotrichum, Acinetobacter and Babesia. The alpha and beta diversity indexes showed a clear separation between the microbiome of high-quality milk and those of the other milk types. Moreover, in the high-quality milk, the relative abundance of Staphylococcus (4.4%), Campylobacter (4.5%), Bacillus (2.5%), Enterococcus (2.4%), Klebsiella (1.3%) and Escherichia (0 .7%) was significantly higher in comparison to other types of milk. On the contrary, the relative abundance of Geotrichum (0.5%) was significantly lower. The microbiome results collected in this study showed significant differences in terms of the relative abundance of bacteria genera, including foodborne pathogen species. These results should be incorporated into risk assessment models tailored to different types of milk.

Keywords: raw milk, foodborne pathogens, microbiome, risk assessment

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Authors: Abu Salim Mustafa

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Water plays a major role in maintaining life on earth, but it can also serve as a matrix for pathogenic organisms, posing substantial health threats to humans. Although, outbreaks of diseases attributable to drinking water may not be common in industrialized countries, they still occur and can lead to serious acute, chronic, or sometimes fatal health consequences. The analysis of drinking water samples from different regions of Kuwait was performed in this study for bacterial and viral contaminations. Drinking tap water samples were collected from 15 different locations of the six Kuwait governorates. All samples were analyzed by confocal microscopy for the presence of bacteria. The samples were cultured in vitro to detect cultivable organisms. DNA was isolated from the cultured organisms and the identity of the bacteria was determined by sequencing the bacterial 16S rRNA genes, followed by BLAST analysis in the database of NCBI, USA. RNA was extracted from water samples and analyzed by real-time PCR for the detection of viruses with potential health risks, i.e. Astrovirus, Enterovirus, Norovirus, Rotavirus, and Hepatitis A. Confocal microscopy showed the presence of bacteria in some water samples. The 16S rRNA gene sequencing of culture grown organisms, followed by BLAST analysis, identified the presence of several non-pathogenic bacterial species. However, one sample had Acinetobacter baumannii, which often causes opportunistic infections in immunocompromised people, but none of the studied viruses could be detected in the drinking water samples analyzed. The results indicate that drinking water samples analyzed from various locations in Kuwait are relatively safe for drinking and do not contain many harmful pathogens.

Keywords: drinking water, microbial contaminant, 16S rDNA, Kuwait

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Authors: Katalin Gombos, Bence Gálik, Krisztina Ildikó Kalács, Krisztina Gödöny, Ákos Várnagy, József Bódis, Attila Gyenesei, Gábor L. Kovács

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Although non-invasive pre-implantation genetic testing for aneuploidy (NIPGT-A) is potentially appropriate to assess chromosomal ploidy of the embryo, practical application of it in a routine IVF center has not been started in the absence of a recommendation. We developed a comprehensive workflow for a clinically applicable strategy for NIPGT-A based on next-generation sequencing (NGS) technology. We performed MALBAC whole genome amplification and NGS on spent blastocyst culture media of Day 3 embryos fertilized with intra-cytoplasmic sperm injection (ICSI). Spent embryonic culture media of morphologically good quality score embryos were enrolled in further analysis with the blank culture media as background control. Chromosomal abnormalities were identified by an optimized bioinformatics pipeline applying a copy number variation (CNV) detecting algorithm. We demonstrate a comprehensive workflow covering both wet- and dry-lab procedures supporting a clinically applicable strategy for NIPGT-A. It can be carried out within 48 h which is critical for the same-cycle blastocyst transfer, but also suitable for “freeze all” and “elective frozen embryo” strategies. The described integrated approach of non-invasive evaluation of embryonic DNA content of the culture media can potentially supplement existing pre-implantation genetic screening methods.

Keywords: next generation sequencing, in vitro fertilization, embryo assessment, non-invasive pre-implantation genetic testing

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Authors: Mina Hojat Ansari, Kamran Bagheri Lankarani, Mohammad Reza Fattahi, Ali Reza Safarpour

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Irritable bowel syndrome (IBS) is a common bowel disorder which is usually diagnosed through the abdominal pain, fecal irregularities and bloating. Alteration in the intestinal microbial composition is implicating to inflammatory and functional bowel disorders which is recently also noted as an IBS feature. Owing to the potential importance of microbiota implication in both efficiencies of the treatment and prevention of the diseases, we examined the association between the intestinal microbiota and different bowel patterns in a cohort of subjects with IBS and healthy controls. Fresh fecal samples were collected from a total of 50 subjects, 30 of whom met the Rome IV criteria for IBS and 20 Healthy control. Total DNA was extracted and library preparation was conducted following the standard protocol for small whole genome sequencing. The pooled libraries sequenced on an Illumina Nextseq platform with a 2 × 150 paired-end read length and obtained sequences were analyzed using several bioinformatics programs. The majority of sequences obtained in the current study assigned to bacteria. However, our finding highlighted the significant microbial taxa variation among the studied groups. The result, therefore, suggests a significant association of the microbiota with symptoms and bowel characteristics in patients with IBS. These alterations in fecal microbiota could be exploited as a biomarker for IBS or its subtypes and suggest the modification of the microbiota might be integrated into prevention and treatment strategies for IBS.

Keywords: irritable bowel syndrome, intestinal microbiota, small whole genome sequencing, fecal samples, Illumina

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Authors: I. Wiedemann, A. R. Sharifi, A. Mählmeyer, C. Knorr

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A requirement for sperm motility is a morphologically intact flagellum with a central axoneme. The flagellar beating is caused by the varying activation and inactivation of dynein molecules which are located in the axoneme. DNAL4 (dynein, axonemal, light chain 4) is regarded as a possible functional candidate gene encoding a small subunit of the dyneins. In the present study, 5814bp of the porcine DNAL4 (GenBank Acc. No. AM284696.1, 6097 bp, 4 exons) were comparatively sequenced using three boars with a high motility (>68%) and three with a low motility (<60%). Primers were self-designed except for those covering exons 1, 2 and 3. Prior to sequencing, the PCR products were purified. Sequencing was performed with an ABI PRISM 3100 Genetic Analyzer using the BigDyeTM Terminator v3.1 Cycle Sequencing Reaction Kit. Finally, 23 SNPs were described and genotyped for 82 AI boars representing the breeds Piétrain, German Large White and German Landrace. The genotypes were used to assess possible associations with standard spermatological parameters (ejaculate volume, density, and sperm motility (undiluted (Motud), 24h (Mot1) and 48h (Mot2) after semen collection) that were regularly recorded on the AI station. The analysis included a total of 8,833 spermatological data sets which ranged from 2 to 295 sets per boar in five years. Only SNP g.1007A>G had a significant effect. Finally, the gene substitution effect using the following statistical model was calculated: Yijk= µ+αi+βj+αβij+b1Sijk+b2Aijk+b3T ijk + b4Vijk+b5(α*A)ijk +b6(β*A)ijk+b7(A*T)ijk+Uijk+eijk where Yijk is the semen characteristics, µ is the general mean, α is the main effect of breed, β is the main effect of season, S is the effect of SNP (g.1007A > G), A is the effect of age at semen collection, V is the effect of diluter, αβ, α*A, β*A, A*T are interactions between the fixed effects, b1-b7 are regression coefficients between y and the respective covariate, U is the random effect of repeated observation on animal and e is the random error. The results from the single marker regression analysis revealed highly significant effects (p < 0.0001) of SNP g.1007A > G on Mot1 resp. on Mot2, resulting in a marked reduction by 11.4% resp. 15.4%. Furthermore a loss of Motud by 4.6% was detected (p < 0.0178). Considering the SNP g.1007A > G as a main factor (dominant-recessive model), significant differences between genotypes AA and AG as well as AA and GG for Mot1 and Mot2 exist. For Motud there was a significant difference between AA and GG.

Keywords: association, DNAL4, porcine, sperm traits

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Authors: Anuj Tyagi, Balwinder Singh, Naveen Kumar B. T., Niraj K. Singh

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Characterization of diverse microbial communities in specific environment plays a crucial role in the better understanding of their functional relationship with the ecosystem. It is now well established that gut microbiome of fish is not the simple replication of microbiota of surrounding local habitat, and extensive species, dietary, physiological and metabolic variations in fishes may have a significant impact on its composition. Moreover, overuse of antibiotics in human, veterinary and aquaculture medicine has led to rapid emergence and propagation of antibiotic resistance genes (ARGs) in the aquatic environment. Microbial communities harboring specific ARGs not only get a preferential edge during selective antibiotic exposure but also possess the significant risk of ARGs transfer to other non-resistance bacteria within the confined environments. This phenomenon may lead to the emergence of habitat-specific microbial resistomes and subsequent emergence of virulent antibiotic-resistant pathogens with severe fish and consumer health consequences. In this study, gut microbiota of freshwater carp (Labeo rohita) was investigated by shotgun metagenomics to understand its taxonomic composition and functional capabilities. Metagenomic DNA, extracted from the fish gut, was subjected to sequencing on Illumina NextSeq to generate paired-end (PE) 2 x 150 bp sequencing reads. After the QC of raw sequencing data by Trimmomatic, taxonomic analysis by Kraken2 taxonomic sequence classification system revealed the presence of 36 phyla, 326 families and 985 genera in the fish gut microbiome. At phylum level, Proteobacteria accounted for more than three-fourths of total bacterial populations followed by Actinobacteria (14%) and Cyanobacteria (3%). Commonly used probiotic bacteria (Bacillus, Lactobacillus, Streptococcus, and Lactococcus) were found to be very less prevalent in fish gut. After sequencing data assembly by MEGAHIT v1.1.2 assembler and PROKKA automated analysis pipeline, pathway analysis revealed the presence of 1,608 Metacyc pathways in the fish gut microbiome. Biosynthesis pathways were found to be the most dominant (51%) followed by degradation (39%), energy-metabolism (4%) and fermentation (2%). Almost one-third (33%) of biosynthesis pathways were involved in the synthesis of secondary metabolites. Metabolic pathways for the biosynthesis of 35 antibiotic types were also present, and these accounted for 5% of overall metabolic pathways in the fish gut microbiome. Fifty-one different types of antibiotic resistance genes (ARGs) belonging to 15 antimicrobial resistance (AMR) gene families and conferring resistance against 24 antibiotic types were detected in fish gut. More than 90% ARGs in fish gut microbiome were against beta-lactams (penicillins, cephalosporins, penems, and monobactams). Resistance against tetracycline, macrolides, fluoroquinolones, and phenicols ranged from 0.7% to 1.3%. Some of the ARGs for multi-drug resistance were also found to be located on sequences of plasmid origin. The presence of pathogenic bacteria and ARGs on plasmid sequences suggested the potential risk due to horizontal gene transfer in the confined gut environment.

Keywords: antibiotic resistance, fish gut, metabolic pathways, microbial diversity

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Authors: Feng Ji Mervin Goh, Edmund Chee Jian Pua, Stuart Alexander Cook

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Dilated cardiomyopathy (DCM) is a common cause of heart failure. Genetic mutations account for 50% of DCM cases with TTN mutations being the most common, accounting for up to 25% of DCM cases. However, the genetic architecture underlying Asian DCM patients is unknown. We evaluated 68 patients (female= 17) with DCM who underwent follow-up at the National Heart Centre, Singapore from 2013 through 2014. Clinical data were obtained and analyzed retrospectively. Genomic DNA was subjected to next-generation targeted sequencing. Nextera Rapid Capture Enrichment was used to capture the exons of a panel of 169 cardiac genes. DNA libraries were sequenced as paired-end 150-bp reads on Illumina MiSeq. Raw sequence reads were processed and analysed using standard bioinformatics techniques. The average age of onset of DCM was 46.1±10.21 years old. The average left ventricular ejection fraction (LVEF), left ventricular diastolic internal diameter (LVIDd), left ventricular systolic internal diameter (LVIDs) were 26.1±11.2%, 6.20±0.83cm, and 5.23±0.92cm respectively. The frequencies of mutations in major DCM-associated genes were as follows TTN (5.88% vs published frequency of 20%), LMNA (4.41% vs 6%), MYH7 (5.88% vs 4%), MYH6 (5.88% vs 4%), and SCN5a (4.41% vs 3%). The average callability at 10 times coverage of each major gene were: TTN (99.7%), LMNA (87.1%), MYH7 (94.8%), MYH6 (95.5%), and SCN5a (94.3%). In conclusion, TTN mutations are not common in Singaporean DCM patients. The frequencies of other major DCM-associated genes are comparable to frequencies published in the current literature.

Keywords: heart failure, dilated cardiomyopathy, genetics, next-generation sequencing

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Authors: Eriko Ochiai, Fumiko Satoh, Keiko Miyashita, Yu Kakimoto, Motoki Osawa

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Sudden and unexpected deaths from unknown causes occur in infants and youths. Recently, molecular links between a part of these deaths and several genetic diseases are examined in the postmortem. For instance, hereditary long QT syndrome and Burgada syndrome are occasionally fatal through critical ventricular tachyarrhythmia. There are a large number of target genes responsible for such diseases, the conventional analysis using the Sanger’s method has been laborious. In this report, we attempted to analyze sudden deaths comprehensively using the next generation sequencing (NGS) technique. Multiplex PCR to subject’s DNA was performed using Ion AmpliSeq Library Kits 2.0 and Ion AmpliSeq Inherited Disease Panel (Life Technologies). After the library was constructed by emulsion PCR, the amplicons were sequenced 500 flows on Ion Personal Genome Machine System (Life Technologies) according to the manufacture instruction. SNPs and indels were analyzed to the sequence reads that were mapped on hg19 of reference sequences. This project has been approved by the ethical committee of Tokai University School of Medicine. As a representative case, the molecular analysis to a 40 years old male who received a diagnosis of Brugada syndrome demonstrated a total of 584 SNPs or indels. Non-synonymous and frameshift nucleotide substitutions were selected in the coding region of heart disease related genes of ANK2, AKAP9, CACNA1C, DSC2, KCNQ1, MYLK, SCN1B, and STARD3. In particular, c.629T-C transition in exon 3 of the SCN1B gene, resulting in a leu210-to-pro (L210P) substitution is predicted “damaging” by the SIFT program. Because the mutation has not been reported, it was unclear if the substitution was pathogenic. Sudden death that failed in determining the cause of death constitutes one of the most important unsolved subjects in forensic pathology. The Ion AmpliSeq Inherited Disease Panel can amplify the exons of 328 genes at one time. We realized the difficulty in selection of the true source from a number of candidates, but postmortem genetic testing using NGS analysis deserves of a diagnostic to date. We now extend this analysis to SIDS suspected subjects and young sudden death victims.

Keywords: postmortem genetic testing, sudden death, SIDS, next generation sequencing

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Authors: Amer A. Hasan, Mehri Igci, Ersin Borazan, Rozhgar A. Khailany, Emine Bayraktar, Ahmet Arslan

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Gastric cancer (GC) has high morbidity and fatality rate in various countries and is still one of the most frequent and deadly diseases. Novel mitogenic and motogenic Gastrokine1 (GKN1) and Gastrokine 2 (GKN2) genes that are highly expressed in the normal stomach epithelium and plays an important role in maintaining the integrity and homeostasis of stomach mucosal epithelial cells. Significant loss of copy number and mRNA transcript of GKN1 and GKN2 gene expression were frequently observed in all types of gastric cancer. In this study, 47 paired samples that were grouped according to the types of gastric cancer and the clinical characteristics of the patients, including gender and average of age were investigated with gene expression analysis and mutation screening by monetering RT-PCR, SSCP and nucleotide sequencing techniques. Both GKN1 and GKN2 genes were observed significantly reduced found by (Wilcoxon signed rank test; p<0.05). As a result of gene screening, no mutation (no different genotype) was detected. It is considered that gene mutations are not the cause of inactivation of gastrokines. In conclusion, the mRNA expression level of GKN1 and GKN2 genes statistically was decreased regardless the gender, age or cancer type of patients. Reduced of gastrokine genes seems to occur at the initial steps of cancer development. In order to understand the investigation between gastric cancer and diagnostic biomarker; further analysis is necessary.

Keywords: gastric cancer, diagnostic biomarker, nucleotide sequencing, semi-quantitative RT-PCR

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Authors: Jiahui Song

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The application of an electric field can cause poration at cell membranes. This includes the outer plasma membrane, as well as the membranes of intracellular organelles. In order to analyze and predict such electroporation effects, it becomes necessary to first evaluate the electric fields and the transmembrane voltages. This information can then be used to assess changes in the pore formation energy that finally yields the pore distributions and their radii based on the Smolchowski equation. The dynamic pore model can be achieved by including a dynamic aspect and a dependence on the pore population density into the pore formation energy equation. These changes make the pore formation energy E(r) self-adjusting in response to pore formation without causing uncontrolled growth and expansion. By using dynamic membrane tension, membrane electroporation in response to a 180kV/cm trapezoidal pulse with a 10 ns on time and 1.5 ns rise- and fall-times is discussed. Poration is predicted to occur at times beyond the peak at around 9.2 ns. Modeling also yields time-dependent distributions of the membrane pore population after multiple pulses. It shows that the pore distribution shifts to larger values of the radius with multiple pulsing. Molecular dynamics (MD) simulations are also carried out for a fixed field of 0.5 V/nm to demonstrate nanopore formation from a microscopic point of view. The result shows that the pore is predicted to be about 0.9 nm in diameter and somewhat narrower at the central point.

Keywords: high-intensity, nanosecond, dynamics, electroporation

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Authors: Yash M. Gupta, Kittisak Buddhachat, Surin Peyachoknagul, Somjit Homchan

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The house cricket, Acheta domesticus is one of the commonly found species of field crickets. Although it is very commonly used as food and feed, the genomic information of house cricket is still missing for genetic investigation. DNA sequencing technology has evolved over the decades, and it has also revolutionized the molecular marker development for genetic analysis. In the present study, we have sequenced the whole genome of A. domesticus using illumina platform based HiSeq X Ten sequencing technology for searching simple sequence repeats (SSRs) in DNA to develop polymorphic microsatellite markers for population genetic analysis. A total of 112,157 SSRs with primer pairs were identified, 91 randomly selected SSRs used to check DNA amplification, of which nine primers were polymorphic. These microsatellite markers have shown cross-amplification with other three species of crickets which are Gryllus bimaculatus, Gryllus testaceus and Brachytrupes portentosus. These nine polymorphic microsatellite markers were used to check genetic variation for forty-five individuals of A. domesticus, Phitsanulok population, Thailand. For nine loci, the number of alleles was ranging from 5 to 15. The observed heterozygosity was ranged from 0.4091 to 0.7556. These microsatellite markers will facilitate population genetic analysis for future studies of A. domesticus populations. Moreover, the transferability of these SSR makers would also enable researchers to conduct genetic studies for other closely related species.

Keywords: cross-amplification, microsatellite markers, observed heterozygosity, population genetic, simple sequence repeats

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Authors: Danyi Wang, Andrew Schriefer, Dennis O'Rourke, Brajendra Kumar, Yang Liu, Fei Zhong, Juergen Scheuenpflug, Zheng Feng

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Purpose: It has been recognized that the microbiome plays critical roles in disease pathogenesis, including cancer, autoimmune disease, and multiple sclerosis. To develop a clinical-grade assay for exploring microbiome-derived clinical biomarkers across disease areas, a two-phase approach is implemented. 1) Identification of the optimal sample preparation reagents using pre-mixed bacteria and healthy donor stool samples coupled with proprietary Sigma-Aldrich® bioinformatics solution. 2) Exploratory analysis of patient samples for enabling precision medicine. Study Procedure: In phase 1 study, we first compared the 16S sequencing results of two ATCC® microbiome standards (MSA 2002 and MSA 2003) across five different extraction kits (Kit A, B, C, D & E). Both microbiome standards samples were extracted in triplicate across all extraction kits. Following isolation, DNA quantity was determined by Qubit assay. DNA quality was assessed to determine purity and to confirm extracted DNA is of high molecular weight. Bacterial 16S ribosomal ribonucleic acid (rRNA) amplicons were generated via amplification of the V3/V4 hypervariable region of the 16S rRNA. Sequencing was performed using a 2x300 bp paired-end configuration on the Illumina MiSeq. Fastq files were analyzed using the Sigma-Aldrich® Microbiome Platform. The Microbiome Platform is a cloud-based service that offers best-in-class 16S-seq and WGS analysis pipelines and databases. The Platform and its methods have been extensively benchmarked using microbiome standards generated internally by MilliporeSigma and other external providers. Data Summary: The DNA yield using the extraction kit D and E is below the limit of detection (100 pg/µl) of Qubit assay as both extraction kits are intended for samples with low bacterial counts. The pre-mixed bacterial pellets at high concentrations with an input of 2 x106 cells for MSA-2002 and 1 x106 cells from MSA-2003 were not compatible with the kits. Among the remaining 3 extraction kits, kit A produced the greatest yield whereas kit B provided the least yield (Kit-A/MSA-2002: 174.25 ± 34.98; Kit-A/MSA-2003: 179.89 ± 30.18; Kit-B/MSA-2002: 27.86 ± 9.35; Kit-B/MSA-2003: 23.14 ± 6.39; Kit-C/MSA-2002: 55.19 ± 10.18; Kit-C/MSA-2003: 35.80 ± 11.41 (Mean ± SD)). Also, kit A produced the greatest yield, whereas kit B provided the least yield. The PCoA 3D visualization of the Weighted Unifrac beta diversity shows that kits A and C cluster closely together while kit B appears as an outlier. The kit A sequencing samples cluster more closely together than both the other kits. The taxonomic profiles of kit B have lower recall when compared to the known mixture profiles indicating that kit B was inefficient at detecting some of the bacteria. Conclusion: Our data demonstrated that the DNA extraction method impacts DNA concentration, purity, and microbial communities detected by next-generation sequencing analysis. Further microbiome analysis performance comparison of using healthy stool samples is underway; also, colorectal cancer patients' samples will be acquired for further explore the clinical utilities. Collectively, our comprehensive qualification approach, including the evaluation of optimal DNA extraction conditions, the inclusion of positive controls, and the implementation of a robust qualified bioinformatics pipeline, assures accurate characterization of the microbiota in a complex matrix for deciphering the deep biology and enabling precision medicine.

Keywords: 16S rRNA sequencing, analytical validation, bioinformatics pipeline, metagenomics

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