Search results for: Illumina DNA sequencing

Authors: M. Sadeghi, H. Pezeshk, R. Tusserkani, A. Sharifi Zarchi, A. Malekpour, M. Foroughmand, S. Goliaei, M. Totonchi, N. Ansari–Pour

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The role and relative importance of intrinsic and extrinsic factors in the development of complex diseases such as cancer still remains a controversial issue. Determining the amount of variation explained by these factors needs experimental data and statistical models. These models are nevertheless based on the occurrence and accumulation of random mutational events during stem cell division, thus rendering cancer development a stochastic outcome. We demonstrate that not only individual genome sequencing is uninformative in determining cancer risk, but also assigning a unique genome sequence to any given individual (healthy or affected) is not meaningful. Current whole-genome sequencing approaches are therefore unlikely to realize the promise of personalized medicine. In conclusion, since genome sequence differs from cell to cell and changes over time, it seems that determining the risk factor of complex diseases based on genome sequence is somewhat unrealistic, and therefore, the resulting data are likely to be inherently uninformative.

Keywords: cancer risk, extrinsic factors, genome sequencing, intrinsic factors

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Authors: Bhaven N. Tandel, Athira Rajeev

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Sequencing batch reactor process is a suspended growth process operating under non-steady state conditions which utilizes a fill and draw reactor with complete mixing during the batch reaction step (after filling) and where the subsequent steps of aeration and clarification occur in the same tank. All sequencing batch reactor systems have five steps in common, which are carried out in sequence as follows, (1) fill (2) react (3) settle (sedimentation/clarification) (4) draw (decant) and (5) idle. The study was carried out in a sequencing batch reactor of dimensions 44cmx30cmx70cm with a working volume of 40 L. Mechanical stirrer of 100 rpm was used to provide continuous mixing in the react period and oxygen was supplied by fish tank aerators. The duration of a complete cycle of sequencing batch reactor was 8 hours. The cycle period was divided into different phases in sequence as follows-0.25 hours fill phase, 6 hours react period, 1 hour settling phase, 0.5 hours decant period and 0.25 hours idle phase. The study consisted of two runs, run 1 and run 2. Run 1 consisted of 6 hours aerobic react period and run 2 consisted of 3 hours aerobic react period followed by 3 hours anoxic react period. The influent wastewater used for the study had COD, BOD, NH3-N and TKN concentrations of 308.03±48.94 mg/L, 100.36±22.05 mg/L, 14.12±1.18 mg/L, and 24.72±2.21 mg/L respectively. Run 1 had an average COD removal efficiency of 41.28%, BOD removal efficiency of 56.25%, NH3-N removal efficiency of 86.19% and TKN removal efficiency of 54.4%. Run 2 had an average COD removal efficiency of 63.19%, BOD removal efficiency of 73.85%, NH3-N removal efficiency of 90.74% and TKN removal efficiency of 65.25%. It was observed that run 2 gave better performance than run 1 in the removal of COD, BOD and TKN.

Keywords: municipal waste water, aerobic, anoxic, sequencing batch reactor

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Authors: Ante Turudic, Filip Varga, Zlatko Liber, Jernej Jakse, Zlatko Satovic, Ivan Radosavljevic, Martina Grdisa

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Microsatellites (SSRs) are highly informative repetitive sequences of 2-6 base pairs, which are the most used molecular markers in assessing the genetic diversity of plant species. Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip) is an outcrossing diploid (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repetitiveness and large size of the genome (haploid genome size of 9,58 pg), previous attempts to develop microsatellite markers using the standard methods were unsuccessful. A next-generation sequencing (NGS) approach was applied on genomic DNA extracted from fresh leaves of Dalmatian pyrethrum. The sequencing was conducted using NovaSeq6000 Illumina sequencer, after which almost 400 million high-quality paired-end reads were obtained, with a read length of 150 base pairs. Short reads were assembled by combining two approaches; (1) de-novo assembly and (2) joining of overlapped pair-end reads. In total, 6.909.675 contigs were obtained, with the contig average length of 249 base pairs. Of the resulting contigs, 31.380 contained one or multiple microsatellite sequences, in total 35.556 microsatellite loci were identified. Out of detected microsatellites, dinucleotide repeats were the most frequent, accounting for more than half of all microsatellites identifies (21,212; 59.7%), followed by trinucleotide repeats (9,204; 25.9%). Tetra-, penta- and hexanucleotides had similar frequency of 1,822 (5.1%), 1,472 (4.1%), and 1,846 (5.2%), respectively. Contigs containing microsatellites were further filtered by SSR pattern type, transposon occurrences, assembly characteristics, GC content, and the number of occurrences against the draft genome of T. cinerariifolium published previously. After the selection process, 50 microsatellite loci were used for primer design. Designed primers were tested on samples from five distinct populations, and 25 of them showed a high degree of polymorphism. The selected loci were then genotyped on 20 samples belonging to one population resulting in 17 microsatellite markers. Availability of codominant SSR markers will significantly improve the knowledge on population genetic diversity and structure as well as complex genetics and biochemistry of this species. Acknowledgment: This work has been fully supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir/ Sch. Bip.) insecticidal potential’ - (PyrDiv) (IP-06-2016-9034).

Keywords: genome assembly, NGS, SSR, Tanacetum cinerariifolium

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Authors: Austin Belfiori, Kevin Rinek, Zach Barcroft, Jennifer Berglind

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Diet influences the composition of the gut microbiota and host's health. Disruption of the balance among the microbiota, epithelial cells, and resident immune cells in the intestine is involved in the pathogenesis of inflammatory bowel disease (IBD). To study the role of gut microbiota in intestinal inflammation, the microbiome of control mice (C57BL6) given a gluten-containing standard diet versus C57BL6 mice given the gluten-free (GF) feed (n=10 in each group) was examined. All mice received the 3% DSS for 5 days. Throughout the study, feces were collected and processed for DNA extraction and MiSeq Illumina sequencing of V4 region of bacterial 16S rRNA gene. Alpha and beta diversities and compositional differences at phylum and genus levels were determined in intestinal microbiota. The mice receiving the GF diet showed a significantly increased abundance of Firmicutes and a decrease of Bacteroides and Lactobacillus at phylum level. Therefore, the gluten free diet led to reductions in beneficial gut bacteria populations. These findings indicate a role of wheat gluten in dysbiosis of the intestinal microbiota.

Keywords: gluten, colitis, microbiota, DSS, dextran sulphate sodium

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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: Darshan Panda, Lambodar Behera, M. J. Baig, Sudhanshu Sekhar

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Low light intensity is a significant limitation for grain yield and quality in rice. However, yield is not significantly reduced in low-light tolerant rice varieties. The work, therefore, planned for comparative transcriptome profiling under low light stress to decipher the genes involved and molecular mechanism of low light tolerance in rice. At the active tillering stage, 50% low light exposure for one day, three days, and five days were given to Swarnaprabha (low light tolerant) and IR8 (low light sensitive) rice varieties. Illumina (HiSeq) platform was used for transcriptome sequencing. A total of 6,652 and 12,042 genes were differentially expressed due to low light intensity in Swarnaprabha and IR8, respectively, as compared to control. CAB, LRP, SBPase, MT15, TF PCL1, and Photosystem I & II complex related gene expressions were mostly increased in Swarnaprabha upon the longer duration of low light exposure, which was not found in IR8 as compared to control. Their expressions were validated by qRT-PCR. The overall study suggested that the maintenance of grain yield in the tolerant variety under low light might be the result of accelerated expression of the genes, which enable the plant to keep the photosynthetic processes moving at the same pace even under low light.

Keywords: rice, low light, photosynthesis, yield

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Authors: Sandisiwe Mangali, Graeme Bradley

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Genome is complete set of the organism's hereditary information encoded as either deoxyribonucleic acid or ribonucleic acid in most viruses. The three different types of genomes are nuclear, mitochondrial and the plastid genome and their sequences which are uncovered by genome sequencing are known as an archive for all genetic information and enable researchers to understand the composition of a genome, regulation of gene expression and also provide information on how the whole genome works. These sequences enable researchers to explore the population structure, genetic variations, and recent demographic events in threatened species. Particularly, genome sequencing refers to a process of figuring out the exact arrangement of the basic nucleotide bases of a genome and the process through which all the afore-mentioned genomes are sequenced is referred to as whole or complete genome sequencing. Gelidium pristoides is South African endemic Rhodophyta species which has been harvested in the Eastern Cape since the 1950s for its high economic value which is one motivation for its sequencing. Its endemism further motivates its sequencing for conservation biology as endemic species are more vulnerable to anthropogenic activities endangering a species. As sequencing, mapping and annotating the Gelidium pristoides genome is the aim of this study. To accomplish this aim, the genomic DNA was extracted and quantified using the Nucleospin Plank Kit, Qubit 2.0 and Nanodrop. Thereafter, the Ion Plus Fragment Library was used for preparation of a 600bp library which was then sequenced through the Ion S5 sequencing platform for two runs. The produced reads were then quality-controlled and assembled through the SPAdes assembler with default parameters and the genome assembly was quality assessed through the QUAST software. From this assembly, the plastid and the mitochondrial genomes were then sampled out using Gelidiales organellar genomes as search queries and ordered according to them using the Geneious software. The Qubit and the Nanodrop instruments revealed an A260/A280 and A230/A260 values of 1.81 and 1.52 respectively. A total of 30792074 reads were obtained and produced a total of 94140 contigs with resulted into a sequence length of 217.06 Mbp with N50 value of 3072 bp and GC content of 41.72%. A total length of 179281bp and 25734 bp was obtained for plastid and mitochondrial respectively. Genomic data allows a clear understanding of the genomic constituent of an organism and is valuable as foundation information for studies of individual genes and resolving the evolutionary relationships between organisms including Rhodophytes and other seaweeds.

Keywords: Gelidium pristoides, genome, genome sequencing and assembly, Ion S5 sequencing platform

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Authors: M. Gogniashvili, I. Maisaia, A. Kotorashvili, N. Kotaria, T. Beridze

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Three types of plasmon (A, B and G) is typical for genus Triticum. In polyploid species - Triticum turgidum L. and Triticum aestivum L. plasmon B is detected. In the forthcoming paper, complete nucleotide sequence of chloroplast DNA of 11 representatives of Georgian wheat polyploid species, carrying plasmon B was determined. Sequencing of chloroplast DNA was performed on an Illumina MiSeq platform. Chloroplast DNA molecules were assembled using the SOAPdenovo computer program. All contigs were aligned to the reference chloroplast genome sequence using BLASTN. For detection of SNPs and Indels and phylogeny tree construction computer programs Mafft and Blast were used. Using Triticum aestivum L. subsp. macha (Dekapr. & Menabde) Mackey var. paleocolchicum Dekapr. et Menabde as a reference, 5 SNPs can be identified in chloroplast DNA of Georgian endemic polyploid wheat. The number of noncoding substitutions is 2, coding substitutions - 3. In comparison with reference DNA two - 38 bp and 56 bp inversions were observed in paleocolchicum subspecies. There were six 1 bp indels detected in Georgian polyploid wheats, all of them at microsatellite stretches. The phylogeny tree shows that subspecies macha, carthlicum and paleocolchicum occupy different positions. According to the simplified scheme based on SNP and indel data, the ancestral, female parent of the all studied polyploid wheat is unknown X predecesor, from which four lines were formed. 1 SNP and two inversions (38 bp and 56 bp) caused the formation of subsp. paleocolchicum. Three other lines are macha, durum and carthlicum lines. Macha line is further divided into two sublines (M_1 and M_4). Carthlicum line includes subsp.carthlicum and T.aestivum - C_1 - C_2 - A_1. One of the central question of wheat domestication is which people(s) participated in wheat domestication? It is proposed that the predecessors of Georgian peoples (Proto-Kartvelians) must be placed, on the evidence of archaic lexical and toponymic data, in the mountainous regions of the western and central part of the Little Caucasus (the Transcaucasian foothills) at least 4,000 years ago. One of the possibility to explain the ‘wheat puzzle’ is that Kartvelian speakers brought domesticated wheat species and subspecis from Fertile Crescent further north to South Caucasus.

Keywords: chloroplast DNA, sequencing, SNP, triticum

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Authors: Anna D. Kotrova, Alexandr N. Shishkin, Elena I. Ermolenko

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The aim. To study the quantitative and qualitative colon bacteria ratio from patients with metabolic syndrome. Materials and methods. Fecal samples from patients of 2 groups were identified and analyzed: the first group was formed by patients with metabolic syndrome, the second one - by healthy individuals. The metagenomics method was used with the analysis of 16S rRNA gene sequences. The libraries of the variable sites (V3 and V4) gene 16S RNA were analyzed using the MiSeq device (Illumina). To prepare the libraries was used the standard recommended by Illumina, a method based on two rounds of PCR. Results. At the phylum level in the microbiota of patients with metabolic syndrome compared to healthy individuals, the proportion of Tenericutes was reduced, the proportion of Actinobacteria was increased. At the genus level, in the group with metabolic syndrome, relative to the second group was increased the proportion of Lachnospira. Conclusion. Changes in the colon bacteria ratio in the gut microbiota of patients with metabolic syndrome were found both at the type and the genus level. In the metabolic syndrome group, there is a decrease in the proportion of bacteria that do not have a cell wall. To confirm the revealed microbiota features in patients with metabolic syndrome, further study with a larger number of samples is required.

Keywords: gut microbiota, metabolic syndrome, metagenomics, tenericutes

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Authors: D. Lepe-Cervantes, E. Leon-Becerril, J. Gomez-Romero, O. Garcia-Depraect, A. Lopez-Lopez

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In this study, raw coffee wastewater (CWW) was used as a complex substrate for anaerobic digestion. The inoculum adaptation stage, microbial diversity analysis and biomethane potential (BMP) tests were performed. A fast inoculum adaptation stage was used by the replacement of vinasse to CWW in an anaerobic sequential batch reactor (AnSBR) operated at mesophilic conditions. Illumina MiSeq sequencing was used to analyze the microbial diversity. While, BMP tests using inoculum adapted to CWW were carried out at different inoculum to substrate (I/S) ratios (2:1, 3:1 and 4:1, on a VS basis). Results show that the adaptability percentage was increased gradually until it reaches the highest theoretical value in a short time of 10 d; with a methane yield of 359.10 NmL CH₄/g COD-removed; Methanobacterium beijingense was the most abundant microbial (75%) and the greatest specific methane production was achieved at I/S ratio 4:1, whereas the lowest was obtained at 2:1, with BMP values of 320 NmL CH₄/g VS and 151 NmL CH₄/g VS, respectively. In conclusion, gradual replacement of substrate was a feasible method to adapt the inoculum in a short time even using complex raw substrates, whereas in the BMP tests, the specific methane production was proportional to the initial amount of inoculum.

Keywords: anaerobic digestion, biomethane potential test, coffee wastewater, fast inoculum adaptation

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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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Authors: Leander Van Neste, Kirk Wojno

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The innate immune system reacts to foreign insult in several unique ways, one of which is phagocytosis of perceived threats such as cancer, bacteria, and viruses. The goal of this study was to look for evidence of phagocytosed RNA from tumor cells in circulating monocytes. While all monocytes possess phagocytic capabilities, the non-classical CD14+/FCGR3A+ monocytes and the intermediate CD14++/FCGR3A+ monocytes most actively remove threatening ‘external’ cellular materials. Purified CD14-positive monocyte samples from fourteen patients recently diagnosed with clinically localized prostate cancer (PCa) were investigated by single-cell RNA sequencing using the 10X Genomics protocol followed by paired-end sequencing on Illumina’s NovaSeq. Similarly, samples were processed and used as controls, i.e., one patient underwent biopsy but was found not to harbor prostate cancer (benign), three young, healthy men, and three men previously diagnosed with prostate cancer that recently underwent (curative) radical prostatectomy (post-RP). Sequencing data were mapped using 10X Genomics’ CellRanger software and viable cells were subsequently identified using CellBender, removing technical artifacts such as doublets and non-cellular RNA. Next, data analysis was performed in R, using the Seurat package. Because the main goal was to identify differences between PCa patients and ‘control’ patients, rather than exploring differences between individual subjects, the individual Seurat objects of all 21 patients were merged into one Seurat object per Seurat’s recommendation. Finally, the single-cell dataset was normalized as a whole prior to further analysis. Cell identity was assessed using the SingleR and cell dex packages. The Monaco Immune Data was selected as the reference dataset, consisting of bulk RNA-seq data of sorted human immune cells. The Monaco classification was supplemented with normalized PCa data obtained from The Cancer Genome Atlas (TCGA), which consists of bulk RNA sequencing data from 499 prostate tumor tissues (including 1 metastatic) and 52 (adjacent) normal prostate tissues. SingleR was subsequently run on the combined immune cell and PCa datasets. As expected, the vast majority of cells were labeled as having a monocytic origin (~90%), with the most noticeable difference being the larger number of intermediate monocytes in the PCa patients (13.6% versus 7.1%; p<.001). In men harboring PCa, 0.60% of all purified monocytes were classified as harboring PCa signals when the TCGA data were included. This was 3-fold, 7.5-fold, and 4-fold higher compared to post-RP, benign, and young men, respectively (all p<.001). In addition, with 7.91%, the number of unclassified cells, i.e., cells with pruned labels due to high uncertainty of the assigned label, was also highest in men with PCa, compared to 3.51%, 2.67%, and 5.51% of cells in post-RP, benign, and young men, respectively (all p<.001). It can be postulated that actively phagocytosing cells are hardest to classify due to their dual immune cell and foreign cell nature. Hence, the higher number of unclassified cells and intermediate monocytes in PCa patients might reflect higher phagocytic activity due to tumor burden. This also illustrates that small numbers (~1%) of circulating peripheral blood monocytes that have interacted with tumor cells might still possess detectable phagocytosed tumor RNA.

Keywords: circulating monocytes, phagocytic cells, prostate cancer, tumor immune response

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Authors: Unchalee Inkampa, Tuanjai Somboonwiwat

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Painting process of automobiles and automobile parts, which is a continuous process based on EDP (Electrode position paint, EDP). Through EDP, all work pieces will be continuously sent to the painting process. Work process can be divided into 2 groups based on the running time: Painting Room 1 and Painting Room 2. This leads to continuous operation. The problem that arises is waiting for workloads onto Painting Room. The grading process EDP to Painting Room is a major problem. Therefore, this paper aim to develop production sequencing method by applying EDP to painting process. It also applied fixed rate launching for painting room and earliest due date (EDD) for EDP process and swap pairwise interchange for waiting time to a minimum of machine. The result found that the developed method could improve painting reduced waiting time, on time delivery, meeting customers wants and improved productivity of painting unit.

Keywords: sequencing, mixed model lines, painting process, electrode position paint

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Authors: Ghorbanali Mohammadi

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New approaches to sequencing mixed-model manufacturing systems are present. These approaches have attracted considerable attention due to their potential to deal with difficult optimization problems. This paper presents Multi-Objective Simulated Annealing Algorithms (MOSAA) approaches to the Just-In-Time (JIT) sequencing problem where workload-smoothing (WL) and the number of set-ups (St) are to be optimized simultaneously. Mixed-model assembly lines are types of production lines where varieties of product models similar in product characteristics are assembled. Moreover, this type of problem is NP-hard. Two annealing methods are proposed to solve the multi-objective problem and find an efficient frontier of all design configurations. The performances of the two methods are tested on several problems from the literature. Experimentation demonstrates the relative desirable performance of the presented methodology.

Keywords: scheduling, just-in-time, mixed-model assembly line, sequencing, simulated annealing

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Authors: Chen Wang, Jared Evans, Yan Asmann

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With the quick evolvement of next-generation sequencing techniques, whole-exome or exome-panel data have become a cost-effective way for detection of small exonic mutations, but there has been a growing desire to accurately detect copy number variations (CNVs) as well. In order to address this research and clinical needs, we developed a sequencing coverage pattern-based method not only for copy number detections, data integrity checks, CNV calling, and visualization reports. The developed methodologies include complete automation to increase usability, genome content-coverage bias correction, CNV segmentation, data quality reports, and publication quality images. Automatic identification and removal of poor quality outlier samples were made automatically. Multiple experimental batches were routinely detected and further reduced for a clean subset of samples before analysis. Algorithm improvements were also made to improve somatic CNV detection as well as germline CNV detection in trio family. Additionally, a set of utilities was included to facilitate users for producing CNV plots in focused genes of interest. We demonstrate the somatic CNV enhancements by accurately detecting CNVs in whole exome-wide data from the cancer genome atlas cancer samples and a lymphoma case study with paired tumor and normal samples. We also showed our efficient reuses of existing exome sequencing data, for improved germline CNV calling in a family of the trio from the phase-III study of 1000 Genome to detect CNVs with various modes of inheritance. The performance of the developed method is evaluated by comparing CNV calling results with results from other orthogonal copy number platforms. Through our case studies, reuses of exome sequencing data for calling CNVs have several noticeable functionalities, including a better quality control for exome sequencing data, improved joint analysis with single nucleotide variant calls, and novel genomic discovery of under-utilized existing whole exome and custom exome panel data.

Keywords: bioinformatics, computational genetics, copy number variations, data reuse, exome sequencing, next generation sequencing

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Authors: Maxime Merheb, Rachel Matar

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Retrieving ancient DNA sequences which in return permit the entire genome sequencing from fossils have extraordinarily improved in recent years, thanks to sequencing technology and other methodological advances. In any case, the quest to search for ancient DNA is still obstructed by the damage inflicted on DNA which accumulates after the death of a living organism. We can characterize this damage into three main categories: (i) Physical abnormalities such as strand breaks which lead to the presence of short DNA fragments. (ii) Modified bases (mainly cytosine deamination) which cause errors in the sequence due to an incorporation of a false nucleotide during DNA amplification. (iii) DNA modifications referred to as blocking lesions, will halt the PCR extension which in return will also affect the amplification and sequencing process. We can clearly see that the issues arising from breakage and coding errors were significantly decreased in recent years. Fast sequencing of short DNA fragments was empowered by platforms for high-throughput sequencing, most of the coding errors were uncovered to be the consequences of cytosine deamination which can be easily removed from the DNA using enzymatic treatment. The methodology to repair DNA sequences is still in development, it can be basically explained by the process of reintroducing cytosine rather than uracil. This technique is thus restricted to amplified DNA molecules. To eliminate any type of damage (particularly those that block PCR) is a process still pending the complete repair methodologies; DNA detection right after extraction is highly needed. Before using any resources into extensive, unreasonable and uncertain repair techniques, it is vital to distinguish between two possible hypotheses; (i) DNA is none existent to be amplified to begin with therefore completely un-repairable, (ii) the DNA is refractory to PCR and it is worth to be repaired and amplified. Hence, it is extremely important to develop a non-enzymatic technique to detect the most degraded DNA.

Keywords: ancient DNA, DNA barcodong, enzymatic repair, PCR

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Authors: Daphne Dean C. Arenos, Glorificacion L. Quinopez

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This study has been conducted to describe the digitized stories authored by a Grade 1 pupil struggling in reading. The main goal was to find out the effect of authoring digital stories on the reading skill of a grade 1 pupil in terms of vocabulary and sequencing skills. To be able to explicate the data collected, a case study approach has been chosen. This case study focused on a 6 years old Filipino child born and raised in Spain and has just transferred to a private school a year ago. The pupil’s struggles in reading, as well as her experiences with digitized stories, were further described. The findings revealed that authoring digital stories facilitate the reading progress of a struggling pupil. The presence of literary elements in the pupil’s stories built her vocabulary and sequencing skills. Hence, authoring digital stories serve as an appropriate and effective scaffold for struggling readers.

Keywords: literary elements, reading skill, scaffold, sequencing skill, vocabulary

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Authors: Negin Parsamanesh, Saman Ameri-Mahabadi, Ali Nikfar, Mojdeh Mansouri, Hossein Chiti, Gita Fatemi Abhari

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Duchenne muscular dystrophy (DMD) is a severe progressive X-linked neuromuscular illness that affects movement through mutations in dystrophin gene. The mutation leads to insufficient, lack of or dysfunction of dystrophin. The cause of DMD was determined in an Iranian family. Exome sequencing was carried out along with a complete physical examination of the family. In silico methods were applied to find the alteration in the protein structure. The homozygous variant in DMD gene (NM-004006.2) was defined as c.2732-2733delTT (p.Phe911CysfsX8) in exon 21. In addition, phylogenetic conservation study of the human dystrophin protein sequence revealed that phenylalanine 911 is one of the evolutionarily conserved amino acids. In conclusion, our study indicated a new deletion in the DMD gene in the affected family. This deletion with an X-linked inheritance pattern is new in Iran. These findings could facilitate genetic counseling for this family and other patients in the future.

Keywords: duchenne muscular dystrophy, whole exome sequencing, iran, metabolic syndrome

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Authors: Borys Stegniy, Anton Gerilovych, Oleksii Solodiankin, Vitaliy Bolotin, Anton Stegniy, Denys Muzyka, Claudio Afonso

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New APMV was isolated from white fronted goose in Ukraine. This isolate was tested serologically using monoclonal antibodies in haemagglutination-inhibition tests against APMV1-9. As the results obtained isolate showed cross reactions with APMV7. Following investigations were provided for the full genome sequencing using random primers and cloning into pCRII-TOPO. Analysis of 100 transformed colonies of E.coli using traditional sequencing gave us possibilities to find only 3 regions, which could identify by BLAST. The first region with the length of 367 bp had 70 % nucleotide sequence identity to the APMV 12 isolate Wigeon/Italy/3920_1/2005 at genome position 2419-2784. Next region (344 bp) had 66 % identity to the same APMV 12 isolate at position 4760-5103. The last region (365 bp) showed 71 % identity to Newcastle disease virus strain M4 at position 12569-12928.

Keywords: APMV, Newcastle disease virus, Ukraine, full genome sequencing

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Authors: Kedibone Masenya, Memory Tekere, Jasper Rees

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Sorghum is an important cereal crop in the world. In particular, it has attracted breeders due to capacity to serve as food, feed, fiber and bioenergy crop. Like any other plant, sorghum hosts a variety of microbes, which can either, have a neutral, negative and positive influence on the plant. In the current study, regions (V3/V4) of 16 S rRNA were targeted to extensively assess bacterial multitrophic interactions in the phyllosphere of sorghum. The results demonstrated that the presence of a pathogen has a significant effect on the endophytic bacterial community. Understanding these interactions is key to develop new strategies for plant protection.

Keywords: bacteria, multitrophic, sorghum, target sequencing

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Authors: Hui Li, Xueying Zhao, Ke Ma, Yu Cao, Fan Yang, Qingwen Xu, Wenbin Liu

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Soil can often link a person or item to a crime scene, which makes it a valuable evidence in forensic casework. Several techniques have been utilized in forensic soil discrimination in previous studies. Because soil contains a vast number of microbiomes, the analyse of soil microbiomes is expected to be a potential way to characterise soil evidence. In this study, we applied massively parallel sequencing (MPS) to soil bacterial profiling on the Ion Torrent Personal Genome Machine (PGM). Soils from different regions were collected repeatedly. V-region 3 and 4 of Bacterial 16S rRNA gene were detected by MPS. Operational taxonomic units (OTU, 97%) were used to analyse soil bacteria. Several bioinformatics methods (PCoA, NMDS, Metastats, LEfse, and Heatmap) were applied in bacterial profiles. Our results demonstrate that MPS can provide a more detailed picture of the soil microbiomes and the composition of soil bacterial components from different region was individualistic. In conclusion, the utility of soil bacterial profiling via MPS of the 16S rRNA gene has potential value in characterising soil evidences and associating them with their place of origin, which can play an important role in forensic science in the future.

Keywords: bacterial profiling, forensic, massively parallel sequencing, soil evidence

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Authors: Ali W. N. Alattabi, Clare B. Harris, Rafid M. Alkhaddar, Montserrat Ortoneda, David A. Phipps, Ali Alzeyadi, Khalid S. Hashim

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This study was performed to optimise the hydraulic retention time (HRT) and study its effects on the sludge characteristics and the effluent quality in an aerobic suspension sequencing batch reactor (ASSBR) treating synthetic wastewater. The results showed that increasing the HRT from 6 h to 12 h significantly improved the COD and Nitrate removal efficiency; it was increased from 78.7% - 75.7% to 94.7% – 97% for COD and Nitrate respectively. However, increasing the HRT from 12 h to 18 h reduced the COD and Nitrate removal efficiency from 94.7% - 97% to 91.1% – 94.4% respectively. Moreover, Increasing the HRT from 18 h to 24 h did not affect the COD and Nitrate removal efficiency. Sludge volume index (SVI) was used to monitor the sludge settling performance. The results showed a direct relationship between the HRT and SVI value. Increasing the HRT from 6 h to 12 h led to decrease the SVI value from 123 ml/g to 82.5 ml/g, and then it remained constant despite of increasing the HRT from 12 h to 18 h and to 24 h. The results obtained from this study showed that the HRT of 12 h was better for COD and Nitrate removal and a good settling performance occurred during that range.

Keywords: COD, hydraulic retention time, nitrate, sequencing batch reactor, sludge characteristics

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Authors: Amin Mojiri, Hamidi Abdul Aziz

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Leachate is created while water penetrates through the waste in a landfill, carrying some forms of pollutants. In literature, for treatment of wastewater and leachate, different ways of biological treatment were used. Sequencing batch reactor (SBR) is a kind of biological treatment. This study investigated the co-treatment of landfill leachate and domestic waste water by SBR and powdered activated carbon augmented (PAC) SBR process. The response surface methodology (RSM) and central composite design (CCD) were employed. The independent variables were aeration rate (L/min), contact time (h), and the ratio of leachate to wastewater mixture (%; v/v)). To perform an adequate analysis of the aerobic process, three dependent parameters, i.e. COD, color, and ammonia-nitrogen (NH3-N or NH4-N) were measured as responses. The findings of the study indicated that the PAC-SBR showed a higher performance in elimination of certain pollutants, in comparison with SBR. With the optimal conditions of aeration rate (0.6 L/min), leachate to waste water ratio (20%), and contact time (10.8 h) for the PAC-SBR, the removal efficiencies for color, NH3-N, and COD were 72.8%, 98.5%, and 65.2%, respectively.

Keywords: co-treatment, landfill Leachate, wastewater, sequencing batch reactor, activate carbon

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Authors: S. Fahiminiya, J. Nadaf, F. Rauch, L. Jerome-Majewska, J. Majewski

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Background: Sequencing of protein coding regions of human genome (Whole Exome Sequencing; WES), has demonstrated a great success in the identification of causal mutations for several rare genetic disorders in human. Generally, most of WES studies have focused on rare variants in coding exons and splicing-sites where missense substitutions lead to the alternation of protein product. Although focusing on this category of variants has revealed the mystery behind many inherited genetic diseases in recent years, a subset of them remained still inconclusive. Here, we present the result of our WES studies where analyzing only rare variants in coding regions was not conclusive but further investigation revealed the involvement of non-coding variants and copy number variations (CNV) in etiology of the diseases. Methods: Whole exome sequencing was performed using our standard protocols at Genome Quebec Innovation Center, Montreal, Canada. All bioinformatics analyses were done using in-house WES pipeline. Results: To date, we successfully identified several disease causing mutations within gene coding regions (e.g. SCARF2: Van den Ende-Gupta syndrome and SNAP29: 22q11.2 deletion syndrome) by using WES. In addition, we showed that variants in non-coding regions and CNV have also important value and should not be ignored and/or filtered out along the way of bioinformatics analysis on WES data. For instance, in patients with osteogenesis imperfecta type V and in patients with glucocorticoid deficiency, we identified variants in 5'UTR, resulting in the production of longer or truncating non-functional proteins. Furthermore, CNVs were identified as the main cause of the diseases in patients with metaphyseal dysplasia with maxillary hypoplasia and brachydactyly and in patients with osteogenesis imperfecta type VII. Conclusions: Our study highlights the importance of considering non-coding variants and CNVs during interpretation of WES data, as they can be the only cause of disease under investigation.

Keywords: whole exome sequencing data, non-coding variants, copy number variations, rare diseases

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Authors: Lien Gysens, Bert Vanmechelen, Maarten Haspeslagh, Piet Maes, Ann Martens

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Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in a clinical presentation on the one hand and the host-dependent clinical outcome of BPV-1 infection, on the other hand, indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of the cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. By generating a significant amount of complete-length BPV genomes, we succeeded in introducing next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

Keywords: Bovine papillomavirus, equine sarcoid, horse, nanopore sequencing, phylogenetic analysis

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Authors: Sunil Dutta

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Synchronization is defined as ‘the sequencing and re-sequencing of all relative and absolute activities in time and space and continuous alignment of those actions with purposeful objective in a complex and dynamic atmosphere. In a complex and dynamic production / maintenance setup, no single group can work in isolation for long. In addition, many activities in projects take place simultaneously at the same time. Work of every section / group is interwoven with work of others. The various activities / interactions which take place in production / overhaul workshops are interlinked because of physical requirements (information, material, workforces, equipment, and space) and dependencies. The activity sequencing is determined by physical dependencies of various department / sections / units (e.g., inventory availability must be ensured before stripping and disassembling of equipment), whereas resource dependencies do not. Theory of constraint facilitates identification, analyses and exploitation of the constraint in methodical manner. These constraints (equipment, manpower, policies etc.) prevent the department / sections / units from getting optimum exploitation of available resources. The significance of theory of constraints for achieving synchronization at overhaul workshop is illustrated in this paper.

Keywords: synchronization, overhaul, throughput, obsolescence, uncertainty

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Authors: Abu S. Mustafa, Mohammad W. Khan, Faraz Shaheed Khan, Nazima Habibi

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Brucellosis is a zoonotic disease of worldwide prevalence. There are at least four species and several strains of Brucella that cause human disease. Brucella genomes have very limited variation across strains, which hinder strain identification using classical molecular techniques, including PCR and 16 S rDNA sequencing. The aim of this study was to perform whole genome sequencing of clinical isolates of Brucella and perform bioinformatics and comparative genomics analyses to determine the existence of genetic differences across the isolates of a single Brucella species and strain. The draft sequence data were generated from 15 clinical isolates of Brucella melitensis (biovar 2 strain 63/9) using MiSeq next generation sequencing platform. The generated reads were used for further assembly and analysis. All the analysis was performed using Bioinformatics work station (8 core i7 processor, 8GB RAM with Bio-Linux operating system). FastQC was used to determine the quality of reads and low quality reads were trimmed or eliminated using Fastx_trimmer. Assembly was done by using Velvet and ABySS softwares. The ordering of assembled contigs was performed by Mauve. An online server RAST was employed to annotate the contigs assembly. Annotated genomes were compared using Mauve and ACT tools. The QC score for DNA sequence data, generated by MiSeq, was higher than 30 for 80% of reads with more than 100x coverage, which suggested that data could be utilized for further analysis. However when analyzed by FastQC, quality of four reads was not good enough for creating a complete genome draft so remaining 11 samples were used for further analysis. The comparative genome analyses showed that despite sharing same gene sets, single nucleotide polymorphisms and insertions/deletions existed across different genomes, which provided a variable extent of diversity to these bacteria. In conclusion, the next generation sequencing, bioinformatics, and comparative genome analysis can be utilized to find variations (point mutations, insertions and deletions) across different genomes of Brucella within a single strain. This information could be useful in surveillance and epidemiological studies supported by Kuwait University Research Sector grants MI04/15 and SRUL02/13.

Keywords: brucella, bioinformatics, comparative genomics, whole genome sequencing

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Authors: Christina Killian, Katie Wall, Séamus Fanning, Guerrino Macori

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Deep-level metagenomics offers a useful technical approach to explore the three dynamic One Health axes: healthcare, domiciliary and veterinary. There is currently limited understanding of the composition of complex biofilms, natural abundance of AMR genes and gene transfer occurrence in these ecological niches. By using a newly established small-scale complex biofilm model, COMBAT has the potential to provide new information on microbial diversity, antimicrobial resistance (AMR)-encoding gene abundance, and their transfer in complex biofilms of importance to these three One Health axes. Shotgun metagenomics has been used to sample the genomes of all microbes comprising the complex communities found in each biofilm source. A comparative analysis between untreated and biocide-treated biofilms is described. The basic steps include the purification of genomic DNA, followed by library preparation, sequencing, and finally, data analysis. The use of long-read sequencing facilitates the completion of metagenome-assembled genomes (MAG). Samples were sequenced using a PromethION platform, and following quality checks, binning methods, and bespoke bioinformatics pipelines, we describe the recovery of individual MAGs to identify mobile gene elements (MGE) and the corresponding AMR genotypes that map to these structures. High-throughput sequencing strategies have been deployed to characterize these communities. Accurately defining the profiles of these niches is an essential step towards elucidating the impact of the microbiota on each niche biofilm environment and their evolution.

Keywords: COMBAT, biofilm, metagenomics, high-throughput sequencing

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Authors: Anyaphat Srithanasuwan, Noppason Pangprasit, Montira Intanon, Phongsakorn Chuammitri, Witaya Suriyasathaporn, Ynte H. Schukken

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Streptococcus uberis is one of the most common mastitis-causing pathogens, with a wide range of intramammary infection (IMI) durations and pathogenicity. This study aimed to compare shared or unique virulence factor gene clusters distinguishing persistent and transient strains of S. uberis. A total of 139 S. uberis strains were isolated from three small-holder dairy herds with a high prevalence of S. uberis mastitis. The duration of IMI was used to categorize bacteria into two groups: transient and persistent strains with an IMI duration of less than 1 month and longer than 2 months, respectively. Six representative S. uberis strains, three from each group (transience and persistence) were selected for analysis. All transient strains exhibited multi-locus sequence types (MLST), indicating a highly diverse population of transient S. uberis. In contrast, MLST of persistent strains was available in an online database (pubMLST). Identification of virulence genes was performed using whole-genome sequencing (WGS) data. Differences in genomic size and number of virulent genes were found. For example, the BCA gene or alpha-c protein and the gene associated with capsule formation (hasAB), found in persistent strains, are important for attachment and invasion, as well as the evasion of the antimicrobial mechanisms and survival persistence, respectively. These findings suggest a genetic-level difference between the two strain types. Consequently, a comprehensive study of 139 S. uberis isolates will be conducted to perform an in-depth genetic assessment through WGS analysis on an Illumina platform.

Keywords: Streptococcus Uberis, mastitis, whole genome sequence, intramammary infection, persistent S. Uberis, transient s. Uberis

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Authors: Pouya Karimi, Ramin Gasemi Shayan, Parsa Sheykhzade

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Ease shotgun DNA sequencing is changing the microbial sciences. Sequencing instruments are compelling to the point that example planning is currently the key constraining element. Here, we present a microfluidic test readiness stage that incorporates the key strides in cells to grouping library test groundwork for up to 96 examples and decreases DNA input prerequisites 100-overlay while keeping up or improving information quality. The universally useful microarchitecture we show bolsters work processes with subjective quantities of response and tidy up or catch steps. By decreasing the example amount necessities, we empowered low-input (∼10,000 cells) entire genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil miniaturized scale settlements with prevalent outcomes. We additionally utilized the upgraded throughput to succession ∼400 clinical Pseudomonas aeruginosa libraries and exhibit magnificent single-nucleotide polymorphism discovery execution that clarified phenotypically watched anti-toxin opposition. Completely coordinated lab-on-chip test arrangement beats specialized boundaries to empower more extensive organization of genomics across numerous fundamental research and translational applications.

Keywords: clinical microbiology, DNA, microbiology, microbial genomics

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