Illumina Paired-end Sequencing Research Articles

Comparative transcriptome analysis between two different cadmium-accumulating genotypes of soybean (Glycine max) in response to cadmium stress

BackgroundCadmium (Cd) is extremely toxic and non-essential for plants. Different soybean varieties differ greatly in their Cd accumulation ability, but little is known about the underlying molecular mechanisms.ResultsHere, we performed transcriptomic analysis using Illumina pair-end sequencing on root tissues from two soybean varieties (su8, high-Cd-accumulating (HAS) and su7, low Cd-accumulating (LAS)) grown with 0 or 50 μM CdSO4. A total of 18.76 million clean reads from the soybean root samples were obtained after quality assessment and data filtering. After Cd treatment, 739 differentially expressed genes (DEGs; 265 up and 474 down) were found in HAS; however, only 259 DEGs (88 up and 171 down) were found in LAS, and 64 genes were same between the two varieties. Pathway enrichment analysis suggested that after cadmium treatment, the DEGs between LAS and HAS were mainly enriched in glutathione metabolism and plant-pathogen interaction pathways. KEGG analysis showed that phenylalanine metabolism responding to cadmium stress in LAS, while ABC transporters responding to cadmium stress in HAS. Besides we found more differential expressed heavy metal transporters such as ABC transporters and zinc transporters in HAS than LAS, and there were more transcription factors differently expressed in HAS than LAS after cadmium treatment in two soybean varieties, eg. bHLH transcription factor, WRKY transcription factor and ZIP transcription factor.ConclusionsFindings from this study will shed new insights on the underlying molecular mechanisms behind the Cd accumulation in soybean.

Complete Plastomes of Ten Rorippa Species (Brassicaceae): Comparative Analysis and Phylogenetic Relationships

The genus Rorippa belongs to the family Brassicaceae, and its members usually have high medicinal value. The genus consists of approximately 75 species and mainly grows in the Northern Hemisphere, occurring in every continent except Antarctica. The taxonomy and phylogenetic relationships of Rorippa are still unsettled, largely due to complex morphological variations in Rorippa, which were caused by frequent hybridization events. Here, we sequenced four complete plastid genomes of Rorippa species by Illumina paired-end sequencing. The four new plastid genomes of Rorippa ranged in total size from 154,671 bp for R. palustris to 154,894 bp for R. sylvestris. There are 130 genes in the four plastomes, embodying 8 rRNA, 37 tRNA, and 85 protein-coding genes. Combining with six published plastid genomes, we carried on comparative and phylogenetic analyses. We found that the ten Rorippa plastid genomes were conservative in gene number and order, total size, genomic structure, codon usage, long repeat sequence, and SSR. Fourteen mutational hotspot regions could be selected as candidate DNA barcoding to distinguish Rorippa plants. The phylogenetic trees clearly identified that ten Rorippa species displayed monophyletic relationships within the tribe Cardamineae based on plastomes and nrDNA ITS sequences. However, there are significant cytonuclear discordances in the interspecific relationships within Rorippa, as well as the intergeneric relationships between Rorippa and its related genera. We inferred that the cytonuclear discordance is most likely a result of interspecific hybridization within Rorippa, as well as intergeneric hybridization with its related genera. These plastid genomes can offer precious information for studies of species authentication, evolutionary history, and the phylogeny of Rorippa.

A Chromosomal-Level Genome of Dermatophagoides farinae, a Common Allergenic Mite Species.

Genome data have been used to find novel allergen from house dust mites. Here, we aim to construct a chromosome-level genome assembly of Dermatophagoides farinae, a common allergenic mite species. We achieved a chromosome-level assembly of D. farinae's genome by integrating PacBio single-molecule real-time sequencing, Illumina paired-end sequencing, and Hi-C technology, followed by annotating allergens and mapping them to specific chromosomes. A 62.43 Mb genome was assembled with a 0.52% heterozygosity rate and a 36.11 Merqury-estimated quality value. The assembled genome represents 92.1% completeness benchmarking universal single-copy orthologs with a scaffold N50 value of 7.11 Mb. Hi-C scaffolding of the genome resulted in construction of 10 pseudochromosomes. The genome comprises 13.01% (7.66 Mb) repetitive sequences and predicts 10,709 protein-coding genes, 96.57% of which are functionally annotated. Moreover, we identified and located 36 allergen groups on specific chromosomes, including allergens Der f 1, Der f 2, Der f 23, Der f 4, Der f 5, Der f 7, and Der f 21 located on chromosomes 2, 1, 7, 3, 4, 6, and 4, respectively. This comprehensive genomic data provides valuable insights into mite biology and evolutionary adaptations, potentially advancing D. farinae allergy research and treatment strategies.

Whole genome analysis reveals unique traits of SARS-CoV-2 in pediatric patients

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to challenge the global healthcare with emerging variants and higher infectivity as well as morbidities. This study investigated potential age-related variations through genomic characterization of the virus under common clinical settings. A cohort comprising 71 SARS-CoV-2 strains from both infected infants and accompanying adults, diagnosed via RT-PCR at a tertiary pediatric hospital and research center, underwent Illumina paired-end sequencing. The subsequent analysis involved standard genomic screening, phylogeny construction, and mutational analyses. The analyzed SARSCoV- 2 strains were compared with globally circulating variants. The overall distribution revealed 67.61 % Delta, 25.7 % Omicron, and 1 % either Kappa or Alpha variants. In 2021, Delta predominated at ∼ 94 %, with Alpha/Kappa accounting for around 5 %. However, in 2022, over 94 % of the samples were Omicron variants, signifying a substantial shift from Delta dominance. Delta variants constituted 69.5 % of infections in adults and 78.5 % in infants, while Omicron variants were responsible for 31 % of cases in infants and 18 % in adults. The Spike region harbored the majority of mutations, with T19R being the most prevalent mutation in the Delta lineage. Notably, the frequencies of this mutation varied between infants and adults. In Omicron samples, G142D emerged as the most prevalent mutation. Our dataset predominantly featured clade 21A and lineage B.1.617.2. This study underscores the differential clinical presentations and genomic characteristics of SARS-CoV-2 in pediatric patients and accompanying adults. Understanding the dynamic evolution of the SARS- CoV-2 in both pediatric and adults can help in strengthening prophylactic measures.

Chloroplast genomes of Caragana tibetica and Caragana turkestanica: structures and comparative analysis

BackgroundThe genus Caragana encompasses multiple plant species that possess medicinal and ecological value. However, some species of Caragana are quite similar in morphology, so identifying species in this genus based on their morphological characteristics is considerably complex. In our research, illumina paired-end sequencing was employed to investigate the genetic organization and structure of Caragana tibetica and Caragana turkestanica, including the previously published chloroplast genome sequence of 7 Caragana plants.ResultsThe lengths of C. tibetica and C. turkestanica chloroplast genomes were 128,433 bp and 129,453 bp, respectively. The absence of inverted repeat sequences in these two species categorizes them under the inverted repeat loss clade (IRLC). They encode 110 and 111 genes (4 /4 rRNA genes, 30 /31tRNA genes, and 76 /76 protein-coding genes), respectively. Comparison of the chloroplast genomes of C. tibetica and C. turkestanica with 7 other Caragana species revealed a high overall sequence similarity. However, some divergence was observed between certain intergenic regions (matK-rbcL, psbD-psbM, atpA-psbI, and etc.). Nucleotide diversity (π) analysis revealed the detection of five highly likely variable regions, namely rps2-atpI, accD-psaI-ycf4, cemA-petA, psbN-psbH and rpoA-rps11. Phylogenetic analysis revealed that C. tibetica’s sister species is Caragana jubata, whereas C. turkestanica’s closest relative is Caragana arborescens.ConclusionsThe present study provides worthwhile information about the chloroplast genomes of C. tibetica and C. turkestanica, which aids in the identification and classification of Caragana species.

Transcriptome analysis of mantle and pearl sac of freshwater pearl mussel Lamellidens marginalis to identify biomineralisation-associated genes

ABSTRACT The freshwater pearl mussel, Lamellidens marginalis, is widely distributed in India and is cultured for freshwater pearl production. Understanding the biomineralisation process sheds light on the genes responsible for nacre secretion. However, information on the transcriptome of L. marginalis mantle tissue and pearl sac is lacking. This study generated transcriptome resources for L. marginalis, identifying genes involved in biomineralisation. Illumina paired-end sequencing yielded 11.13 million raw reads, assembled into 133,246 contigs and 26,373 unigenes. Of these, 21,033 unigenes exhibited homology with previously reported molluscan proteins. Genes related to biomineralisation, such as pif, perlucin, calreticulin, calmodulin, chitin synthase, chitin dehydrogenase, carbonic anhydrase, tyrosinase, shell matrix protein, dermatopontin isoform X1, chitinase 3, chitinase domain-containing protein 1, and putative chitinase-1were identified. This baseline information will help to compare the expression levels between the phenotypes of high-quality and low-quality pearls. Further investigations, incorporating large sample sizes and phenotype information, could assist in identifying type I genetic markers linked to pearl quality.

Genome mining of Escherichia coli WG5D from drinking water source: unraveling antibiotic resistance genes, virulence factors, and pathogenicity

BackgroundEscherichia coli, a ubiquitous inhabitant of the gut microbiota, has been recognized as an indicator of fecal contamination and a potential reservoir for antibiotic resistance genes. Its prevalence in drinking water sources raises concerns about the potential dissemination of antibiotic resistance within aquatic ecosystems and the subsequent impact on public health. The ability of E. coli to acquire and transfer resistance genes, coupled with the constant exposure to low levels of antibiotics in the environment, underscores the need for comprehensive surveillance and rigorous antimicrobial stewardship strategies to safeguard the quality and safety of drinking water supplies, ultimately mitigating the escalation of antibiotic resistance and its implications for human well-being.MethodsWG5D strain, isolated from a drinking water distribution source in North-West Province, South Africa, underwent genomic analysis following isolation on nutrient agar, anaerobic cultivation, and DNA extraction. Paired-end Illumina sequencing with a Nextera XT Library Preparation kit was performed. The assembly, annotation, and subsequent genomic analyses, including phylogenetic analysis using TYGS, pairwise comparisons, and determination of genes related to antimicrobial resistance and virulence, were carried out following standard protocols and tools, ensuring comprehensive insights into the strain’s genomic features.ResultsThis study explores the notable characteristics of E. coli strain WG5D. This strain stands out because it possesses multiple antibiotic resistance genes, encompassing tetracycline, cephalosporin, vancomycin, and aminoglycoside resistances. Additionally, virulence-associated genes indicate potential heightened pathogenicity, complemented by the identification of mobile genetic elements that underscore its adaptability. The intriguing possibility of bacteriophage involvement and factors contributing to pathogenicity further enriches our understanding. We identified E. coli WG5D as a potential human pathogen associated with a drinking water source in South Africa. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. It further identified WG5D as a potential human pathogen. The occurrence of E. coli WG5D raised the awareness of the potential pathogens and the carrying of antibiotic resistance in drinking water.ConclusionsThe findings of this study have highlighted the advantages of the genomic approach in identifying the bacterial species and antibiotic resistance genes of E. coli and its potential as a human pathogen.

Genomic insights into local-scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages.

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovars A-C, is the leading infectious cause of blindness worldwide. Africa bears the highest burden, accounting for over 86 % of global trachoma cases. We investigated Ct serovar A (SvA) and B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and the implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swabs across nine Gambian villages during a 6 month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted whole-genome sequencing (WGS) on a limited number (n=43) of Ct-positive samples with an omcB load ≥10 from four villages. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had highest identity to ompA from strain A/HAR13 (NC_007429) and 30 had highest identity to ompA from strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains, Ct A/HAR13 and Ct B/Jali20, indicated an single nucleotide polymorphism accumulation rate of 2.4×10-5 per site per year for the Gambian SvA and 1.3×10-5 per site per year for SvB variants (P<0.0001). Variant calling resulted in a total of 1371 single nucleotide variants (SNVs) with a frequency >25 % in SvA sequences, and 438 SNVs in SvB sequences. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP) and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB, hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

The complete chloroplast genome and phylogenetic analysis of Bauhinia glauca subsp. hupehana (Craib) T. C. Chen 1988

Bauhinia glauca subsp. hupehana (Craib) T. C. Chen 1988, a member of the Leguminosae family, Cercidoideae subfamily, and Bauhinia genus, has a rich history of traditional usage in Chinese medicine. Renowned for its analgesic properties, it is commonly employed for managing inflammation and pain. This study aimed to sequence the complete chloroplast genome of B. glauca subsp. hupehana using Illumina paired-end sequencing data. The chloroplast genome spans 156,967 bp and consists of four main regions: the large single-copy (LSC) region (89,185 bp), the small single-copy (SSC) region (19,146 bp), and a pair of inverted repeats (IRs) (24,318 bp). The overall GC content of the chloroplast genome is 36.19%, with specific values of 33.99%, 29.79%, and 42.76% for the LSC, SSC, and IR regions, respectively. A total of 128 genes were annotated in the chloroplast genome, including 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis revealed that B. glauca subsp. hupehana is closely related to Bauhinia racemose, indicating a sister taxon relationship between the two species. This study significantly contributes to the chloroplast genomic resource for Bauhinia, laying the groundwork for future phylogenetic investigations within the genus.

Exploring the use of metabarcoding to reveal eukaryotic associations with mononchids nematodes.

Nematodes play a vital ecological role in soil and marine ecosystems, but there is limited information about their dietary diversity and feeding habits. Due to methodological challenges, the available information is based on inference rather than confirmed observations. The lack of correct dietary requirements also hampers rearing experiments. To achieve insight into the prey of mononchid nematodes, this study employed high-throughput Illumina paired-end sequencing using universal eukaryotic species 18S primers on 10 pooled mononchid nematode species, namely Mylonchulus brachyuris, M. brevicaudatus, Mylonchulus sp., Clarkus parvus, Prionchulus sp. M. hawaiiensis, M. sigmaturellus, M. vulvapapillatus, Anatonchus sp. and Miconchus sp. The results indicate that mononchids are associated with a remarkable diversity of eukaryotes, including fungi, algae, and protists. While the metabarcoding approach, first introduced here for mononchids, proved to be a simple and rapid method, it has several limitations and crucial methodological challenges that should be addressed in future studies. Ultimately, such methods should be able to evaluate the dietary complexity of nematodes and provide a valuable avenue for unraveling the dietary requirements of previously unculturable nematodes. This can contribute to the methodology of understanding their feeding habits and contributions to ecosystem dynamics.

Chloroplast genome of Ocimum basilicum var. purpurascens Bentham 1830 (Lamiaceae)

Ocimum basilicum var. purpurascens Bentham 1830 (Red Rubin Basil) is an aromatic herb belonging to the family Lamiaceae and is known for its medicinal uses. It is commonly used in traditional medicine to treat cardiovascular diseases and obesity. It possesses anti-inflammatory, antioxidant, antifungal, and anti-spasmodic properties. In our recent study, we assembled the chloroplast genome sequence of O. basilicum var. purpurascens using Illumina paired-end sequencing technology. The assembled chloroplast genome was 152,407 base pairs (bp), inclusive of a large single-copy (LSC) region accounting for 83,409 bp and a small single-copy (SSC) region spanning 17,604 bp. Two inverted repeats (IRs) interspersed these regions, each 25,697 bp long. The chloroplast genome harbored 132 genes, comprising 88 protein-coding genes, 36 transfer RNA (tRNA), and eight rRNA genes. Among these, nine genes encompassed a single intron, two presented with two introns, with the remaining devoid of any introns. The overall GC content of the chloroplast genome was determined to be 38%. The GC content in the LSC, SSC, and IR regions was 35.9%, 31.6%, and 43.1%, respectively. Our phylogenetic exploration of the chloroplast genomes elucidated that O. basilicum var. purpurascens exhibits close genetic affinity with O. basilicum var. basilicum and other constituents of the Ocimum genus within the Lamiaceae family.

Chromosome-scale Genome assembly of the critically endangered White-eared Night-Heron (Gorsachius magnificus)

The White-eared Night-Heron (Gorsachius magnificus, G. magnificus) is a critically endangered heron that is very poorly known and only found in southern China and northern Vietnam, with an estimated population of 250 to 999 mature individuals. However, the lack of a reference genome has hindered the implementation of conservation management efforts. In this study, we present the first high-quality chromosome-scale reference genome, which was assembled by integrating PacBio long-reads sequencing, Illumina paired-end sequencing, and Hi-C technology. The genome has a total length of 1.176 Gb, with a scaffold N50 of 84.77 Mb and a contig N50 of 18.46 Mb. Utilizing Hi-C data, we anchored 99.89% of the scaffold sequences onto 29 pairs of chromosomes. Additionally, we identified 18,062 protein-coding genes in the genome, with 95.00% of which were functionally annotated. Notably, BUSCO assessment confirmed the presence of 97.2% of highly conserved Aves genes within the genome. This chromosome-level genome assembly and annotation will be valuable for future investigating the G. magnificus’s evolutionary adaptation and conservation.

Complete chloroplast genome of Artabotrys hexapetalus (L.f.) Bhandari 1965 (Annonaceae)

Artabotrys hexapetalus (L.f.) Bhandari, 1965, an evergreen climbing shrub of significant value, is prominent in Chinese history and culture. The whole-gene sequencing of its chloroplast genome using Illumina pair-end sequencing data is conducted during this research. The complete chloroplast genome was determined to be 178,457 bp in size, separated by a large single copy (LSC) and a small single copy (SSC) region of 90,803 and 3,066 bp, respectively. A total of 134 genes were identified, including 90 protein-coding genes, 36 tRNA, and eight rRNA genes. Phylogenetic analysis revealed a close relationship between A. hexapetalus and Artabotrys pilosus, forming a sister branch with 100% support. The study suggests that the chloroplast genome of A. hexapetalus provides valuable insights into its evolutionary history and will contribute to the conservation efforts of this species.

Genomic exploration of Sesuvium sesuvioides: comparative study and phylogenetic analysis within the order Caryophyllales from Cholistan desert, Pakistan

BackgroundThe Aizoaceae family’s Sesuvium sesuvioides (Fenzl) Verdc is a medicinal species of the Cholistan desert, Pakistan. The purpose of this study was to determine the genomic features and phylogenetic position of the Sesuvium genus in the Aizoaceae family. We used the Illumina HiSeq2500 and paired-end sequencing to publish the complete chloroplast sequence of S. sesuvioides.ResultsThe 155,849 bp length cp genome sequence of S. sesuvioides has a 36.8% GC content. The Leucine codon has the greatest codon use (10.6%), 81 simple sequence repetitions of 19 kinds, and 79 oligonucleotide repeats. We investigated the phylogeny of the order Caryophyllales’ 27 species from 23 families and 25 distinct genera. The maximum likelihood tree indicated Sesuvium as a monophyletic genus, and sister to Tetragonia. A comparison of S. sesuvioides, with Sesuvium portulacastrum, Mesembryanthemum crystallinum, Mesembryanthemum cordifolium, and Tetragonia tetragonoides was performed using the NCBI platform. In the comparative investigation of genomes, all five genera revealed comparable cp genome structure, gene number and composition. All five species lacked the rps15 gene and the rpl2 intron. In most comparisons with S. sesuvioides, transition substitutions (Ts) were more frequent than transversion substitutions (Tv), producing Ts/Tv ratios larger than one, and the Ka/Ks ratio was lower than one. We determined ten highly polymorphic regions, comprising rpl22, rpl32-trnL-UAG, trnD-GUC-trnY-GUA, trnE-UUC-trnT-GGU, trnK-UUU-rps16, trnM-CAU-atpE, trnH-GUG-psbA, psaJ-rpl33, rps4-trnT-UGU, and trnF-GAA-ndhJ.ConclusionThe whole S. sesuvioides chloroplast will be examined as a resource for in-depth taxonomic research of the genus when more Sesuvium and Aizoaceae species are sequenced in the future. The chloroplast genomes of the Aizoaceae family are well preserved, with little alterations, indicating the family’s monophyletic origin. This study’s highly polymorphic regions could be utilized to build realistic and low-cost molecular markers for resolving taxonomic discrepancies, new species identification, and finding evolutionary links among Aizoaceae species. To properly comprehend the evolution of the Aizoaceae family, further species need to be sequenced.

Characterization of the Cambaroides wladiwostokiensis Birstein & Vinogradov, 1934 (Decapoda: Astacidea) Mitochondrial Genome Using Genome Skimming and the Phylogenetic Implications within the Astacidea Infraorder.

The mitochondrial genome is a powerful tool for exploring and confirming species identity and understanding evolutionary trajectories. The genus Cambaroides, which consists of freshwater crayfish, is recognized for its evolutionary and morphological complexities. However, comprehensive genetic and mitogenomic data on species within this genus, such as C. wladiwostokiensis, remain scarce, thereby necessitating an in-depth mitogenomic exploration to decipher its evolutionary position and validate its species identity. The mitochondrial genome of C. wladiwostokiensis was obtained through shallow Illumina paired-end sequencing of total DNA, followed by hybrid assembly using both de novo and reference-based techniques. Comparative analysis was performed using available Cambaroides mitochondrial genomes obtained from National Center for Biotechnology Information (NCBI). Additionally, phylogenetic analyses of 23 representatives from three families within the Astacidea infraorder were employed using the PhyloSuite platform for sequence management and phylogenetic preparation, to elucidate phylogenetic relationships via Bayesian Inference (BI), based on concatenated mitochondrial fragments. The resulting genome, which spans 16,391 base pairs was investigated, revealing 13 protein-coding genes, rRNAs (12S and 16S), 19 tRNAs, and a putative control region. Comparative analysis together with five other Cambaroides mitogenomes retrieved from GenBank unveiled regions that remained unread due to challenges associated with the genome skimming technique. Protein-coding genes varied in size and typically exhibited common start (ATG) and stop (TAA) codons. However, exceptions were noted in ND5 (start codon: GTG) and ND1 (stop codon: TAG). Landscape analysis was used to explore sequence variation across the five available mitochondrial genomes of Cambaroides. Collectively, these findings reveal variable sites and contribute to a deeper understanding of the genetic diversity in this genus alongside the further development of species-specific primers for noninvasive monitoring techniques. The partitioned phylogenetic analysis of Astacidea revealed a paraphyletic origin of Asian cambarids, which confirms the data in recent studies based on both multilocus analyses and integrative approaches.

Antimicrobial Resistance in the Upper Respiratory Tract of Children Compared with Adults

Abstract Background Antimicrobial resistance (AMR) is a growing global public health threat. In 2019, resistant bacterial infections and respiratory tract infections were a leading cause of death in children worldwide. While the gut is a known reservoir of AMR genes, there is little data on the AMR burden in the respiratory tract. We used metagenomic RNA next-generation sequencing (mNGS) to describe bacterial AMR genes detected in the upper respiratory tract of children and compare with adults. Methods We leveraged two established cohort studies of children and adults diagnosed with acute respiratory illnesses in the inpatient and outpatient setting in California and Colorado. Nasopharyngeal (NP) swabs were collected between March to September 2020. Specimens underwent RNA extraction and paired-end Illumina sequencing. AMR genes were detected using the ARG-ANNOT database and the CZID pipeline. Children ≤18 years of age and adults≥ 40 years of age were eligible for inclusion in the analysis. We described and compared AMR genes and AMR gene classes detected in children and adults. P-values were calculated using the Wilcoxon rank-sum test. A multiple logistic regression model was fitted to evaluate the association of age cohort (children vs adults) with presence of AMR genes, while accounting for possible confounding from sex and encounter setting. 95% confidence intervals (CI) were calculated using the Wald CI. Results NP swab mNGS data from 236 patients (82 children, 154 adults) were analyzed. Children were a median age of 4 years (range: 0-17 years), and adults were a median age of 62 years (range: 40-89 years). Both cohorts were 50% male. AMR genes were detected in 44 (54%) children and 109 (71%) adults. Genes conferring resistance to beta-lactams (n=112, 47%), macrolides (n=89, 38%), and tetracyclines (n=72, 31%) were the most frequently detected. Among children, children ages 0 to 2 years had a higher number of AMR genes than children of other age groups, particularly when compared with children ages 3 to 10 years (p-value: 0.031) (Figure 1A). When comparing children with adults, children had fewer AMR genes (p-value: 0.014) as well as fewer AMR gene classes (p-value: 0.031) (Figure 1B). In the multiple logistic regression model, children still had lower odds of any AMR genes detected compared with adults (odds ratio: 0.39, 95% CI: 0.21-0.71). Figure 1. Box plots of (A) the number of antimicrobial resistance (AMR) genes detected in children stratified by age, and (B) the number of AMR genes and AMR gene classes detected in children (n=82) and adults (n=154). The size of the individual data points within the box plots correlates with the number of patients at each data point. Conclusion Children had fewer AMR genes and gene classes than adults in their upper respiratory tract microbiome. Despite this, over half of the children had detectable resistance genes, and children 2 years and younger had more AMR genes than other children. Almost half of the children had resistance genes to beta-lactams, the most frequently prescribed antibiotics to children. These findings suggest that presence of AMR organisms in the airway is common even in children, and that continued efforts to reduce AMR burden in this population are essential.

Application of Deep Sequencing in Phage Display.

This chapter describes the workflow to implement deep sequencing into standard phage display experiments on protein libraries. By harvesting the power of high throughput of these techniques, it allows for comprehensive analysis of the naïve library and library evolution in response to selection by ligand binding. The mutagenized target region of the protein variants encoded by the phage pool is analyzed by Illumina paired-end sequencing. Sequence data are processed to extract selection-enriched amino acid motifs. In addition, a complementary long-read sequencing approach is proposed enabling the monitoring of display vector stability.

Genome Assembly and Microsatellite Marker Development Using Illumina and PacBio Sequencing in the Carex pumila (Cyperaceae) from Korea.

This study is the first to report the characterization of Carex pumila genomic information. Assembly of the genome generated a draft of C. pumila based on PacBio Sequel II and Illumina paired-end sequencing, which was assembled from 2941 contigs with an estimated genome size of 0.346 Gb. The estimate of repeats in the genome was 31.0%, and heterozygosity ranged from 0.426 to 0.441%. The integrity evaluation of the assembly revealed 1481 complete benchmarked universal single-copy orthologs (BUSCO) (91.76%), indicating the high quality of the draft assembly. A total of 23,402 protein-coding genes were successfully predicted and annotated in the protein database. UpsetR plots showed that 7481 orthogroups were shared by all species. The phylogenetic tree showed that C. pumila is a close but distant relative of Ananas comosus. C. pumila had greater contraction (3154) than expansion (392). Among the extended gene families, aquaporins have been found to be enriched. Primers for microsatellite markers determined 30 polymorphic markers out of 100. The average number of alleles amplified by these 30 polymorphic markers was 4 to 12, with an average polymorphism information content (PIC) value of 0.660. In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of C. pumila.

Differential gene expression between viruliferous and non-viruliferous Schizaphis graminum (Rondani)

An experiment was performed to measure the effect of Cereal Yellow-Dwarf Virus (CYDV), strain CYDV-RPV, on gene expression in its insect vector, greenbug aphid (Schizaphis graminum (Rondani)). RNA was sampled in three replicates from four treatments (biotypes B and H with or without carried CYDV), at 0, 1, 2, 3, 5, 10, 15 and 20 days from the introduction of carrier and virus-free greenbugs to uninfected wheat cv. ‘Newton’. Illumina paired-end sequencing produced 1,840,820,000,000 raw reads that yielded 1,089,950,000 clean reads, which were aligned to two greenbug, Trinity transcriptome assemblies with bowtie2. Read counts to contigs were analyzed with principal components and with DESeq2 after removing contaminating contigs of wheat or microbial origin. Likelihood ratio tests with one transcriptome showed that CYDV influenced gene expression about seven-fold less than time or biotype, which were approximately equal. With the other transcriptome, virus, time, and biotype were about equally important. Pairwise comparisons of virus to no virus for each timepoint yielded estimates of fold-change that comprised expression profiles for each contig when ordered by timepoint. Hierarchical clustering separated expression profiles into 20 groups of contigs that were significantly differentially expressed for at least one timepoint. Contigs were also sorted by timepoint of maximally differential expression between virus and no virus. All contigs that were significantly differentially expressed at FDR = 0.05 were annotated by blast searches against NCBI nr and nt databases. Interesting examples of up-regulation with virus included a lysosomal-trafficking regulator, peptidylprolylisomerase, RNA helicase, and two secreted effector proteins. However, carried virus did not consistently change aphid gene expression overall. Instead there was complex interaction of time, biotype, host response, and virus.

Comparison of chloroplast genomes and phylogenetic analysis of four species in Quercus section Cyclobalanopsis

The identification in Quercus L. species was considered to be difficult all the time. The fundamental phylogenies of Quercus have already been discussed by morphological and molecular means. However, the morphological characteristics of some Quercus groups may not be consistent with the molecular results (such as the group Helferiana), which may lead to blurring of species relationships and prevent further evolutionary researches. To understand the interspecific relationships and phylogenetic positions, we sequenced and assembled the CPGs (160,715 bp-160842 bp) of four Quercus section Cyclobalanopsis species by Illumina pair-end sequencing. The genomic structure, GC content, and IR/SC boundaries exhibited significant conservatism. Six highly variable hotspots were detected in comparison analysis, among which rpoC1, clpP and ycf1 could be used as molecular markers. Besides, two genes (petA, ycf2) were detected to be under positive selection pressure. The phylogenetic analysis showed: Trigonobalanus genus and Fagus genus located at the base of the phylogeny tree; The Quercus genus species were distincted to two clades, including five sections. All Compound Trichome Base species clustered into a single branch, which was in accordance with the results of the morphological studies. But neither of group Gilva nor group Helferiana had formed a monophyly. Six Compound Trichome Base species gathered together in pairs to form three branch respectively (Quercus kerrii and Quercus chungii; Quercus austrocochinchinensis with Quercus gilva; Quercus helferiana and Quercus rex). Due to a low support rate (0.338) in the phylogeny tree, the interspecies relationship between the two branches differentiated by this node remained unclear. We believe that Q. helferiana and Q. kerrii can exist as independent species due to their distance in the phylogeny tree. Our study provided genetic information in Quercus genus, which could be applied to further studies in taxonomy and phylogenetics.