Illumina HiSeq X10 Research Articles

UV-B Halotolerant Bacteria from Marakkanam Saltpan and Biology of UV-B Tolerant Pontibacillus salipaludis Based on Whole Genome Sequencing

Saltpans in South India experience intense sunlight and ultraviolet radiation (UV). Therefore, it was hypothesized that halophilic bacteria living there could tolerate UV radiation. To test this, sediment and water samples from the Marakkanam Saltpan were collected and exposed to an artificial UV-B lamp. After irradiation, an aliquot of the sample was spread on nutrient agar (prepared in sterilized source water), and two bacterial strains were obtained, named SBO and SBY. Based on 16S rRNA gene phylogeny, these strains were identified as Bacillus sp. and Pontibacillus salipaludis, respectively. Another aliquot of the UV-B-exposed sample was inoculated into the nutrient broth (in sterilized source water), and incubated for a week in the dark. Later, it was subjected to Illumina MiSeq-based sequencing for the V3-V4 region (16S rRNA gene). Results revealed the presence of 13 bacterial phyla. However, the phylum Bacillota (48.59%) and Pseudomonadota (27.36%) accounted for more than 75% of the diversity. Pontibacillus was the most abundant genus, accounting for 44% of total bacterial diversity. The whole genome of P. salipaludis was sequenced on Illumina HiSeqX to decipher possible genes and pathways involved in UV-B tolerance. The genome size was estimated to be 4.05 MB, with a mean G + C content of 40.82%. RASTtk-based genome annotation revealed 4217 coding sequences and 78 RNAs. Related to the UV-B tolerance, sixteen genes involved in carotenoid and prodigiosin biosynthetic pathways were found. antiSMASH showed the presence of terpene-type carotenoids. More than sixty genes involved in various DNA repair pathways were found.

Comparative genomics and characterization of a multidrug-resistant Acinetobacter baumannii VRL-M19 isolated from a crowded setting in India

A crowded vegetable market serves as a mass gathering, posing a potential risk for infection transmission. In this study, we isolated a multidrug-resistant Acinetobacter baumannii strain, VRL-M19, from the air of such a market and conducted comparative genomics and phenotypic characterization. Antimicrobial susceptibility testing, genome sequencing using Illumina HiSeq X10, and pan-genome analysis with 788 clinical isolates identified core, accessory, and unique drug-resistant determinants. Mutational analysis of drug-resistance genes, virulence factor annotation, in vitro pathogenicity assessment, subsystem analysis, Multilocus sequence typing, and whole genome phylogenetic analysis were performed. VRL-M19 exhibited multidrug resistance with 69 determinants, and analysis across 788 clinical isolates and 350 Indian isolates revealed more accessory genes (52 out of 69) in the Indian isolates. Multiple mutations were observed in drug target modification genes, and the strain was identified as a moderate biofilm-former with 55 virulence factors. Whole genome phylogenetics indicated a close relationship between VRL-M19 and clinical A. baumannii strains. In conclusion, our comprehensive study suggests that VRL-M19 is a multidrug-resistant, potential pathogen with biofilm-forming capabilities, closely associated with clinical A. baumannii strains.

Immune and metabolic perturbations in COVID-19-associated pulmonary mucormycosis: A transcriptome analysis of innate immune cells.

The mechanisms underlying COVID-19-associated pulmonary mucormycosis (CAPM) remain unclear. We use a transcriptomic analysis of the innate immune cells to investigate the host immune and metabolic response pathways in patients with CAPM. We enrolled subjects with CAPM (n = 5), pulmonary mucormycosis (PM) without COVID-19 (n = 5), COVID-19 (without mucormycosis, n = 5), healthy controls (n = 5) without comorbid illness and negative for SARS-CoV-2. Peripheral blood samples from cases were collected before initiating antifungal therapy, and neutrophils and monocytes were isolated. RNA sequencing was performed using Illumina HiSeqX from monocytes and neutrophils. Raw reads were aligned with HISAT-2 pipeline and DESeq2 was used for differential gene expression. Gene ontology (GO) and metabolic pathway analysis were performed using Shiny GO application and R packages (ggplot2, Pathview). The derangement of core immune and metabolic responses in CAPM patients was noted. Pattern recognition receptors, dectin-2, MCL, FcRγ receptors and CLEC-2, were upregulated, but signalling pathways such as JAK-STAT, IL-17 and CARD-9 were downregulated; mTOR and MAP-kinase signalling were elevated in monocytes from CAPM patients. The complement receptors, NETosis, and pro-inflammatory responses, such as S100A8/A9, lipocalin and MMP9, were elevated. The major metabolic pathways of glucose metabolism-glycolysis/gluconeogenesis, pentose phosphate pathway, HIF signalling and iron metabolism-ferroptosis were also upregulated in CAPM. We identified significant alterations in the metabolic pathways possibly leading to cellular iron overload and a hyperglycaemic state. Immune responses revealed altered recognition, signalling, effector functions and a pro-inflammatory state in monocytes and neutrophils from CAPM patients.

The complete chloroplast genome of Porella gracillima Mitt. (Porellaceae) and phylogenetic relationships to other bryophytes

Porella gracillima Mitt. (Jungermanniidae, Porellaceae), a bryophyte is widespread in temperate Asia and North America. In Korea, P. gracillima is mainly observed in shaded and dried rocks or tree trunks on mountains. Here, we determined the complete chloroplast (cp) genome sequence of P. gracillima to provide useful genetic information in the phylogenetic relationship, phylogeographic history, and conservation of the species. The complete cp genome of P. gracillima was assembled using NGS Illumina HiSeqX platform. The cp genome was 121,867 bp in length (GC contents, 33.7%) and showed a typical quadripartite structure, consisting of a large single copy (LSC) of 83,406 bp, a small single copy (SSC) of 19,692 bp, and two inverted repeats (IRs) of 9,385 bp. Phylogenetic analysis shows that Porellaceae was a sister group of Radulaceae, which agrees with the findings of the previous phylogenetic studies. Our cp genome data of P. gracillima may contribute to a better understanding of the evolution of the Porella in Porellaceae and will help to infer its molecular identification, thereby providing a guideline for conservation.

Changes in the Abundances of Cervical and Rectal Mycobiota during Chemoradiotherapy in Cervical Cancer

Globally, cervical cancer is the fourth most frequent cancer in women. The local and gut microbiomes of cervical cancer patients primarily consist of bacteria, viruses, and fungi. Bacterial composition has been previously associated with response to chemoradiotherapy (CRT) and patient outcome. Recently, our group has demonstrated that the HPV virome dynamically shifts during treatment and was associated with treatment response. Although connections between fungi and cervicovaginal health have been established, little is known about the fungal microbiome during treatment of cervical cancer. In this study, we sought to explore changes in fungal distribution throughout CRT for a cohort of cervical cancer patients. This study includes 57 patients diagnosed with cervical cancer at a single institution with samples collected throughout CRT timepoints: baseline, week 1, week 3, and week 5. 170 swab specimens were included in this analysis: 138 cervical swabs from 56 patients and 32 rectal swabs from 9 patients. Whole genome sequencing data was obtained from the swabs using the Illumina HiSeqX platform (2 × 150bp). Fungal reads were log transformed to reduce variability and skewness and normalized to the total library size resulting in log normalized fungal reads per million (RPM). Bacterial reads were normalized with the same methodology. Timepoint analysis was performed using Wilcoxon signed rank tests or Friedman tests (with Dunn's multiple comparisons test) when comparing two or more time points, respectively. All swab samples contained reads mapped to fungi. Of the 3.70 × 109 total reads across all sequenced samples, 19.2% did not map specifically to the human genome: 91.8% of these non-human reads could not be mapped to an individual microbial genome. Of the remaining 8.2% of non-human reads that mapped to a specific microbial genome (1.6% of total), 97.4% were classified as bacterial (1.5% of total), 0.71% as virus (0.011% of total), and 0.44% as fungal (0.0069% of total). Comparison of fungal RPM in the cervical and rectal microbiome revealed a significant decrease during treatment from baseline to week 5 (cervical, P = 0.0002; rectal, P = 0.0273). Distribution of bacterial reads exhibited similar trends as decreases were observed between baseline and week 5 for both cervical (P = 0.0116) and rectal (P = 0.0195) samples. Fungi to bacteria distribution ratios in the rectal microbiome revealed significant differences when comparing across all timepoints (P = 0.0041), baseline vs. week 1 (P = 0.0028), baseline vs. week 3 (P = 0.0389), and baseline vs. week 5 (P = 0.0113) with all three later timepoints higher relative to baseline. The distributions of cervical and gut fungal reads and their relationship to the bacteriome shift during CRT. Further investigation into characterizing fungi and their relationship with other microbiota will be valuable to understanding its potential associations with cervical cancer and treatment response.

Low-coverage whole-genome sequencing for the effective diagnosis of early endometrial cancer: A pilot study

BackgroundEndometrial carcinoma (EC) is a disease that predominantly affects peri- and post-menopausal women and its incidence has continued to rise over recent years. Since the gold standard for EC diagnosis—hysteroscopic biopsy—is invasive, expensive, and unsuitable for wide use, there is an urgent need for a non-invasive method that exhibits both high sensitivity and high specificity. We therefore investigated the efficacy of UterCAD (the uterine exfoliated cell chromosomal aneuploidy detector) using tampon-collected specimens for the early detection of EC. MethodsWe prospectively recruited 51 patients with a history of abnormal bleeding and who planned to undergo hysteroscopic examination or hysterectomy between March 2020 and January 2021. Before executing an invasive procedure, a tampon was inserted into the patient's vagina for 6 h to collect exfoliated cells from the uterine cavity. Total DNA was extracted and low-coverage whole-genome sequencing was performed on an Illumina HiSeq X10, and we analyzed the differences in chromosomal status between women with EC and those bearing benign lesions using UterCAD. ResultsThirty EC patients—including 26 with endometrioid carcinoma (EEC) and four with uterine serous carcinoma (USC), as well as 14 benign cases—were enrolled in our final analysis. Copy-number variations (CNVs) were detected in tampon specimens collected from 26 EC patients (83.3%), including 21 with EEC (80.7%) and four with USC (100%). In the benign group, only one woman with focal atypical hyperplasia presented with a 10q chromosomal gain (P < 0.001). In the EC group, the most common CNVs were copy gains of 8q (N = 14), 2q (N = 4), and 10q (N = 3); and copy losses of 2q (N = 3) and 17p (N = 2). When we stratified by FIGO stage, the CNV rates in stages IA, IB, and II/III were 83.3% (15/18), 85.7% (6/7), and 80.0% (4/5), respectively. At the optimal cutoff (|Z| ≥ 2.3), UterCAD discriminated 83.3% of EC cases from benign cases, with a specificity of 92.9%. ConclusionsWe initially reported that UterCAD could serve as a non-invasive method for the early detection of EC, especially in the rare and aggressive USC subtype. The use of UterCAD might thus avoid unnecessary invasive procedures and thereby reduce the treatment burden on patients.

Fate of antimicrobial resistance genes (ARG) and ARG carriers in struvite production process from human urine

Struvite, a human urine-derived fertilizer types, is characterized by its low water solubility that renders it a slow-releasing eco-friendly fertilizer. Knowing the fate of antibiotic resistance genes in struvite is important since human urine carries microorganisms, viruses and mobilomes. In this study, urine samples were collected and struvite production was done using MgCl2. From the fresh, stored urine and struvite, DNA was extracted and metagenomic sequencing was done using Illumina HiSeqX. Metagenome-derived genome sequence analysis revealed the dominance of phages of Streptococcus, Bacillus and Escherichia, with nearly 50% abundance of streptococcus phage in fresh urine. Increased antibiotic resistance genes were found in the stored urine than in fresh and struvite samples. The top five resistance genes in all the three samples were to aminoglycosides, carbapenem, chloramphenicol, erythromycin and efflux pump, with key carrying pathogens including Acinetobacter, Aeromonas and Enterococcus. The identified families for carbapenem, aminoglycoside resistance and efflux pump were shown persistent in struvite with a shift in gene families. The detection of resistance-gene-laden mobilomes, including the last-resort antibiotics in the struvite sample, requires due attention before the implementation of struvite as fertilizer. Further optimization of the struvite production process with regard to the minimization of mobilomes is recommended.

Decolorization of p-nitrophenol and draft genome sequence of Pseudomonas sp. strain PNPG3: A preliminary report

Aim: To study decolorization (with concomitant depletion) of p-nitrophenol by a bacterial strain designated as PNPG3 and determination of draft genome sequence of the strain to understand its potential. Methodology: A comparative study of PNP’s decolorization (with concomitant removal) in three different test conditions was undertaken. The experiment was carried out in one-liter volume Schott Duran bottles. Genomic DNA was extracted and draft genome sequence was determined using Illumina HiSeqX platform. Raw reads were assembled and subjected to subsystem classification using several bioinformatics tools (RAST, PATRIC, and NCBI’s PGAP pipelines). The genome sequence was deposited at the NCBI Genome database and the strain PNPG3 was also deposited at MTCC, IMTECH, Chandigarh. Results: The bacterial strain PNPG3 could carry out decolorization with concomitant removal of PNP in all three sets of experiments, including one set, where only distilled water was used. The best decolorization (with concomitant PNP removal) capacity was recorded for set D (with Minimal Medium, MM; PNP, and free cells) followed by set E (MM, PNP, and immobilized cells) and set B (distilled water, PNP and free cells) conditions. The size of the draft genome sequence of the strain PNPG3 was 6,566,321 bp, with 62.26% GC contents. The genome had 6210 protein-coding sequences and 66 tRNA genes. The predictive presence of different types of proteases and siderophore receptors indicated its possible potential for industrial applications and plant growth-promoting activities. Interpretation: The bacterium Pseudomonas sp. strain PNPG3 has the capacity to decolorize p-nitrophenol even in presence of distilled water and it remains viable for up to twelve days. The genome sequence revealed that the strain harbored genes responsible for the metabolism of aromatic compounds, chemotaxis, protease, and siderophore receptors indicating the versatile nature of the strain. Key words: Decolorization, P-nitrophenol, Pseudomonas sp., RAST, Xenobiotic

Genome resources and whole genome resequencing of Phytophthora rubi isolates from red raspberry.

Phytophthora rubi is a primary causal agent of Phytophthora root rot and wilting of raspberry (Rubus idaeus L.) worldwide. The disease is a major concern for raspberry growers in Canada and USA. To date, no information is available on genomic diversity of P. rubi population from raspberry in Canada. Using a PCR-free library prep with dual-indexing for an Illumina HiSEQX running a 2x150 bp configuration, we generated whole genome sequence data of P. rubi isolates (n = 25) recovered during 2018 to 2020 from nine fields, four locations and four cultivars of raspberry growing areas of British Columbia, Canada. The assembled genome of 24 isolates of P. rubi averaged 8,541 scaffolds, 309× coverage, and 65,960,000 bp. We exploited single nucleotide polymorphisms (SNPs) obtained from whole genome sequence data to analyze the genome structure and genetic diversity of the P. rubi isolates. Low heterozygosity among the 72% of pathogen isolates and standardized index of association revealed that those isolates were clonal. Principal component analysis, discriminant analysis of principal component, and phylogenetic tree revealed that P. rubi isolates clustered with the raspberry specific cultivars. This study provides novel resources and insight into genome structure, genetic diversity, and reproductive biology of P rubi isolated from red raspberry. The availability of the P. rubi genomes also provides valuable resources for future comparative genomic and evolutionary studies for oomycetes pathogens.

A chromosome scale assembly of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), genome

ObjectiveThe tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is a pest damaging many cultivated crops in North America. Although partial transcriptome data are available for this pest, a genome assembly was not available for this species. This assembly of a high-quality chromosome-length genome of TPB is aimed to develop the genetic resources that can provide the foundation required for advancing research on this species.ResultsThe initial genome of TPB assembled with paired-end nucleotide sequences generated with Illumina technology was scaffolded with Illumina HiseqX reads generated from a proximity ligated (HiC) library to obtain a high-quality genome assembly. The final assembly contained 3963 scaffolds longer than 1 kbp to yield a genome of 599.96 Mbp. The N50 of the TPB genome assembly was 35.64 Mbp and 98.68% of the genome was assembled into 17 scaffolds larger than 1 Mbp. This megabase scaffold number is the same as the number of chromosomes observed in karyotyping of this insect. The TPB genome is known to have high repetitive DNA content, and the reduced assembled genome size compared to flowcytometric estimates of approximately 860 Mbp may be due to the collapsed assembly of highly similar regions.

Analysis of prevailing pertussis strains from different regions in China after the application of acellular vaccines.

Analysis of prevailing pertussis strains from different regions in China after the application of acellular vaccines.

P024 Genetic determinants of antifungal drug resistance in Fusarium solani species complex

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMDue to its challenging diagnosis and treatment, fungal keratitis is one of the most serious kinds of corneal infection. The Fusarium solani species complex is responsible for nearly half of all fungal keratitis cases. Fusarium infection is difficult to treat because of the increased antifungal resistance of Fusarium species.ObjectiveTo check the antifungal susceptibility in keratitis-causing isolates of F. solani species complex.To find the genetic determinants of resistance using whole genome sequencing.MethodologyPrior to AFST and according to CLSI, clinical isolates of Fusarium species (n = 5) were cultured on potato dextrose agar for 7 days at room temperature. Fungal spores were harvested using 0.8% NaCl and antifungal drug susceptibility testing of 6 different antifungal drugs were tested using CLSI standards broth microdilution method and minimum inhibitory concentrations were obtained. After 24 and 48 h, broth microdilution plates were manually examined. The wells for growth control were also examined. The minimum inhibitory concentration of anti-fungal drugs was the lowest dose that inhibits growth completely (compared to the growth control well). Minimal effective concentration (MEC) was also determined against antifungal drugs. Whole genome sequencing was done using Illumina Hiseqx10. Bioinformatics analysis was done using different bioinformatics tools and software (FASTQC, SPAdes, OmicsBox, AUGUSTUS, etc.).ResultsIn this study the antifungal drug susceptibility from average MIC value were found as fluconazole (512 μg/ml) > itraconazole (32 μg/ml) > amphotericin B (8 μg/ml) > natamycin (8 μg/ml) > posaconazole (2 μg/ml) > voriconazole (1 μg/ml). Fluconazole had a higher MIC value (512 g/ml) in all isolates, however, voriconazole was shown to be more sensitive, with a lower MIC range (0.25-4 g/ml). Glyoxalase/Bleomycin resistance protein, mfs—multidrug resistance transporter, fusaric acid resistance protein, efflux pump antibiotic resistance protein, and copper resistance protein were found by genome-based analysis.ConclusionAs a conclusion of this study, we observed antifungal drug resistance in Fusarium spp., which is often used to treat keratitis in patients, employing fluconazole, itraconazole, and amphotericin B. Resistance to first-line azoles was as a gene variant which contributed to antifungal drug resistance. This study using the phenotypic and genotypic characterization of drug resistance patterns will help to combat antifungal drug resistance.

First De novo whole genome sequencing and assembly of mutant Dendrobium hybrid cultivar 'Emma White'.

The Dendrobium hybrid cultivar 'Emma White' is an ornamental, successfully commercialised orchid. We used a gamma ray-induced early flowering mutant and the Illumina HiSeqX10 sequencing platform to generate the first draft de novo whole genome sequence and assembly. The draft sequence was 678,650,699bp in length, comprising 447,500 contigs with an N50 of 1423 and 33.48% GC content. Comparing 95,529 predicted genes against the Uniprot database revealed 60,741 potential genes governing molecular functions, biological processes and cellular components. We identified 216,232 simple sequence repeats and 138,856 microsatellite markers. Chromosome-level genome assembly of Dendrobium huoshanense was used to RagTag-scaffold available contigs of the mutant, revealing a total length of 687,254,899bp with an N50 of 2096. The longest final contiguous length was 18,000,059bp from 30,571bp. BUSCO genome completeness was 93.6%. This study is valuable for investigating the mechanisms of mutation, and developing Dendrobium hybrid cultivars using mutation breeding.

First Report of Apple rubodvirus 2 Infecting Pear (Pyrus communis) in South Africa.

Apple rubbery wood virus 2 (ARWV-2; Rott et al., 2018) belong to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD) that is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes and increased cold sensitivity (Jakovljevic et al., 2017, Rott et al., 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al., 2017; Rott et al., 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. Forelle, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms, except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (one gram) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7371 nts, M was 1289 nts and S was 1463 nts in length, generated with 7341, 626 and 9161 reads for segment L, M and S, respectively. Segment S had the highest read coverage (524.87x), followed by segment L (88.07x) and M (36.60x). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M) and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100%, 80.92% and 100% of these accessions of segments L, M and S, respectively and were deposited in GenBank as accessions MZ593725- MZ593727. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R) and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al., 2018). Amplicon sequences (510 bp (L), 645 bp (M) and 799 bp (S)) were confirmed with bi-directional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay as mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars Bosc (22 samples), Abate (10 samples), Rosemarie (3 samples), Forelle (9 samples), Packham's Triumph (12 samples) and Early Bon Chretien (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.

First Report of Coguvirus eburi Infecting Pear (Pyrus communis) in South Africa.

Coguvirus eburi is a member of the genus Coguvirus in the family Phenuviridae (Khun et al., 2020). The species Coguvirus eburi was established to include citrus virus A (CiVA), which is a negative-sense, single-stranded RNA virus that was first found infecting sweet orange in southern Italy via high-throughput sequencing (HTS) (Navarro et al., 2018). This virus was also found to infect pome fruits in France, such as pear (Svanella-Dumas et al., 2019). More recently CiVA infections have been associated with impietratura disease in citrus (Beris et al. 2021). In the summer of 2021, leaf samples were collected from a pear tree (Pyrus communis cv. Bosc, B175) in the Koue Bokkeveld, South Africa as part of a virus survey. Sample B175 displayed no visual disease symptoms. One gram of leaf petioles was used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). Ribo-depleted RNA was prepared (Ribo-Zero Plant kit) and a sequencing library constructed (Illumina TruSeq Stranded Total RNA). The RNA library was paired-end (2 × 100 bp) sequenced on an Illumina HiSeqX instrument (Macrogen, South Korea). A total of 47,750,152 reads were obtained. Raw data was trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly performed with CLC Genomics Workbench 11.0.1 (Qiagen) (Default parameters) using high quality reads yielded 75250 contigs. BLASTn analysis identified two viral contigs with high nucleotide (nt) identity to apple stem pitting virus (ASPV) and CiVA. The CiVA contig was 9400 nts and on closer examination, a concatemer of CiVA RNA1 and RNA2. The concatenation occurred due to the characteristic near-identical nucleotides shared at the 5' and 3' ends of RNA1 and RNA2 of these negative-stranded RNA viruses (Navarro et al., 2018). After splitting and curation, the RNA1 contig was 6664 nts and the RNA2 contig 2686 nts. A total of 51397 and 34820 reads were used to construct these contigs resulting in an average depth of coverage of 761 and 1281 for RNA1 and RNA2, respectively. The contigs had the highest nt identity to the complete CiVA GenBank accessions MT720885.1 (95.53%) and MW148460.1 (96.03%), spanning 99.6% and 98.1 % of the genomes of RNA1 and RNA2, respectively. These contigs were submitted as partial genomes to GenBank as accessions MZ463039 and MZ463040. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of CiVA in sample B175. Two RT-PCR assays, directed at RNA1 and RNA2 respectively (Bester et al. (2021)) were used to generate amplicons. Amplicon sequences were confirmed with bi-directional Sanger sequencing. Twenty-one additional samples from the same orchard as B175 as well as other samples from the Koue Bokkeveld and Elgin areas, including cultivars Abate (10 samples), Forelle (10 samples), Early Bon Chretien (3 samples), Packham's Triumph (12 samples) and Rosemarie (3 samples), were all surveyed for CiVA using the same RT-PCR assays as mentioned above. Thirty-six of the 59 samples tested were positive for CiVA, which further confirms the presence and wide-spread distribution of this virus in the limited survey conducted in pears in South Africa. However, no association with any disease symptoms or specific cultivar were identified. This is the first report of CiVA infecting pear in South Africa. This study therefore contributed to investigating the distribution of this virus and will assist the South African plant material certification scheme to assess the incidence of CiVA in South Africa.

Detection of Gene Mutations in Patients with Clinically Suspected Heritary Spherocytosis By Next Generation Sequencing

Introduction: Hereditary spherocytosis (HS) is the most frequent form of inherited hemolytic anemia characterized by the presence of spherical-shaped erythrocytes on the peripheral blood smear, hemolysis, splenomegaly, jaundice, and gallstones. The clinical severity of HS varied widely, and its prevalence is 1 in 2,000 in Northern European population and1 in 100,000 in China. The diagnosis can be based on the physical examination, complete red blood cell count, reticulocytes count, medical history and specific tests, preferentially the EMA test test and AGLT, as well as molecular diagnosis particularly gene sequencing. To date, mutations in at least five genes (ANK1, EPB42, SLC4A1, SPTA1, and SPTB) are associated with different subtypes of HS. Next-generation gene sequencing (NGS) is a high-throughput parallel sequencing method and its high-sensitivity is widely used in variation identification in clinical settings with a wide range of human diseases including inherited disorders. This study is aim to discover pathogenic mutations in clinically suspected HS patients by NGS to confirm and/or discriminate diagnosis.Methods: A cohort of seven patients with clinically suspected HS were enrolled in this study. The DNA samples were extracted from peripheral blood samples of the patients and their parents (if available), and were sheared into 200-300bp fragments using the Covaris ultrasonic instrument, followed by whole-exome sequencing. NGS samples were sequenced on the Illumina HiSeq X10 instrument according to manufacturer's protocol. Raw data analysis was performed with the Illumina Pipeline software and the identified mutations were confirmed by Sanger sequencing. Sequences were aligned to the hg19 human genome reference using information from databases including NCBI dbSNP, HapMap, and the 1000 Human Genomes Project dataset. SOAPsnp and Samtools were used to locate the mutations, and the potential functional impacts of the identified variants were assessed using SIFT, PolyPhen-2 and MutationTaster software.Results: Seven patients with age from 8-44yrs were enrolled in our cohort study. NGS results have identified 4 missense mutation SLC4A1 (P854L), SLC4A1 (P455R), SEC23B (P25H and P567R), 3 nonsense mutation ANK1 (R1334X), SPTB (R1756X), SPTB (E1602X), and 1 splicing mutation SPTB (c.5937+1G>C) in seven patients respectively. Three novel mutations, SPTB (R1756X and E1602X) that were stop-gain single nucleotide variants (SNVs) and splicing variant SPTB (c.5937+1G>C) were identified responsible for hereditaryspherocytosis (HS). Considering that the missense mutation SLC4A1 (P854L) was inherited from asymptomatic mother, the pathogenicity of this site is low. Moreover, the mutation SPTB (E1602X) is a de novo mutation and thus likely to be pathogenic. However, the compound heterozygous mutation SEC23B (P25H and P567R) is responsible for congenital dyserythropoietic anemia (CDA) which represent similar clinical symptoms with HS, may have been misdiagnosed and needs to be further studied.Conclusions: Together, we have identified three novel mutations, two stop-gain mutation SPTB (R1756X and E1602X) and one splicing variant SPTB (c.5937+1G>C), in hereditary spherocytosis patients. Our results suggested that genetic diagnosis by NGS is a fast, economic and accurate way to detect and identify pathogenic alterations of inherited diseases like hereditary spherocytosis, and more importantly a useful tool for differential diagnosis. DisclosuresNo relevant conflicts of interest to declare.

Draft Genome Sequence of Pandrug-Resistant Pseudomonas aeruginosa SPA03, Isolated from a Patient with Benign Prostatic Hyperplasia.

ABSTRACTThe draft genome of pandrug-resistant Pseudomonas aeruginosa strain SPA03, which belongs to global high-risk sequence type 357 (ST357) and was isolated from a patient with benign prostatic hyperplasia, is presented in this report. The genome assembly was generated by combining short-read Illumina HiSeq-X Ten and long-read Oxford Nanopore Technologies MinION sequence data using the Unicycler assembler.

Comparative analysis of the ovarian transcriptome reveals novel insights into fertility differences in Large White sows.

Fertility is the most important economic trait in sows, as it is critical for profitability. Considerable phenotypic variation in litter size exists in Large White sows. However, relatively little is known about the underlying molecular and genetic bases. An experiment was conducted to screen key genes that affect the fecundity of pigs during the luteal (L) and follicular phases (F) of the estrous cycle. Eight sows (n = 4 for high fertility sows and n = 4 for low fertility sows) were sacrificed on day 14 (day 1 = first day of estrus) after estrus in the L phase. Another eight sows were slaughtered on day 20 of the estrous cycle in the F phase. Sixteen ovarian tissue samples were collected at the different sacrifice time points. Total RNA extracted was used to construct the library and then sequence on an Illumina HiSeq X10 system. Differentially expressed genes (DEGs) between high and low fertility in Large White sows were identified, and their potential biological functions were analyzed using bioinformatics analysis. In total, 457 DEGs (161 up-regulated and 296 down-regulated genes) were detected in the ovarian tissues of the high and low fertility groups in the L phase of the estrous cycle. Furthermore, 475 DEGs (253 up-regulated and 222 down-regulated genes) were identified in the F phase. Twenty-nine DEGs were common to both comparisons. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the DEGs were mainly associated with steroid biosynthesis, the Hippo signaling pathway, and lysosomes. Others, such as MSMO1, CYP27B1, and CTSB, were related to reproduction. These results will contribute to a better understanding of the individual differences in fertility at the transcriptome level, which may provide useful information to explore new ways to improve fertility in pigs.

Non-invasive detection of urothelial carcinoma (UC) by cost-effective low-coverage whole genome sequencing from urine exfoliated cells DNA.

1552 Background: Urothelial carcinoma (UC) is a malignancy with frequent chromosomal aberrations. The FISH assays were more sensitive as compared to cytology tests. Here we investigated cost-effective whole genome sequencing technology, which is able to detect all chromosomal aberrations for UC diagnoses. Methods: UC patients and control group are prospectively recruited in trial NCT03998371. First-morning-voided urine were freshly collected before TURBT or cystectomy. Urine Exfoliated Cells DNA was analyzed by illumina HiSeq X10, followed by genotyping by bioinformatics workflow UCAD. Results: 195 individuals were prospectively recruited. 121 UC patients and 67 non-tumor diseases were included in this study. 7 other malignancies as confirmed by pathological testing were excluded. Frequent chromosome copy number changes were found in cancer patients as compared non-tumor controls, including chromosome 3 gain, 17 gain, 7 gain and 9p loss used in FISH assays were found. In addition to that, chr9q loss, 8q gain, 5q loss, 17p loss, 11p loss, 1q gain, 8p loss, 10q loss, 6q loss, 4q loss and 11q loss were also frequent in cancer patients (AUC &gt; 0.65). Metacentric chromosomes showed better AUC compared to acrocentric and telocentric chromosomes (P = 1.7e-03). A novel diagnosis model UCAD was built by incorporating all the chromosomal changes. The model reached performance of AUC = 0.933. At the optimal cutoff |Z| &gt; = 3.16, the sensitivity, specificity and accuracy were 84.7%, 97.9% and 89.0% respectively. The prediction positivity was found correlated with urine microscopy visible epithelial cells (P = 0.00069), tumor invasiveness (Ta/Tis vs the other, P = 0.0048) and tumor grade (P = 0.0030), but not microscopy RBC/WBC findings, urine culture findings, smoke and drinking history. The UCAD model outperformed cytology tests by predicting all 16-cytology positive and 12 cytology negative tumors with comparable specificity. The model found 75.0% more tumors. And UCAD identified more upper urinary tract cancer (P = 0.012) and smaller tumors ( &lt; 3cm, P = 5.9e-04). The adding of cytology to UCAD did not improve diagnosing sensitivity and specificity. UCAD reproduce the diagnoses among morning - void urine, morning, afternoon urine samples with correlation coefficient R2&gt; 0.98. All the urine samples showed high concordances with matched tumor samples (R2&gt; 0.85). Conclusions: UCAD could be a high specific, robust UC diagnoses method with improved sensitivity as compared to cytology tests. Clinical trial information: NCT03998371.

Gene panel for Mendelian strokes

BackgroundMendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke. Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate...