cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

The lineage of coronavirus SARS-CoV-2 of Russian origin: Genetic characteristics and correlations with clinical parameters and severity of coronavirus infection

The identification of new SARS-CoV-2 and human protein and gene targets, which may be markers of the severity and outcome of the disease, are extremely important during the COVID-19 pandemic. The goal of this study was to carry out genetic analysis of SARS-CoV-2 RNA samples to elucidate correlations of genetic parameters (SNPs) with clinical data and severity of COVID-19 infection.Material and Methods. The study included viral RNA samples isolated from 56 patients with COVID-19 infection who received treatment at the City Hospital No. 40 of St. Petersburg from 04/18/2020 to 04/18/2021. Patients underwent physical examination with the assessments of hemodynamic and respiratory parameters, clinical risk according to National Early Warning Score (NEWS), computed tomography (CT) of the chest, and laboratory studies including clinical blood analysis, assessment of ferritin, C-reactive protein (CRP), interleukin-6 (IL-6), lactate dehydrogenase (LDH), D-dimer, creatinine, and glucose levels. All patients tested positive for SARS-CoV-2 RNA by polymerase chain reaction (PCR). Single nucleotide polymorphisms (SNPs) in viral RNA were identified through the creation of cDNA libraries by targeted sequencing (MiSeq Illumina). Bioinformatic analysis of viral samples was performed using the viralrecon v2 pipeline with the further annotation via Pangolin and Nextlade. Sampled genomes were visualized using the Integrative Genomics Viewer (IGV) software. Statistical data processing (descriptive statistics and graphical analysis of data relationships from diff erent tables) was performed using a GraphPad device on the Prism 8.01 platform.Results. A comparative analysis of SNP frequencies in the virus genome in samples from deceased and discharged patients was carried out. The SNPs associated with risk of death (OR > 1), neutral SNPs (OR = 1), and protective SNPs (OR < 1) were identifi ed. Patient samples were infected with 14 lines of SARS-CoV-2, fi ve of which (B.1.1.129, B.1.1.407, B.1.1.373, B.1.1.397, and B.1.1.152) were of Russian origin. The SNPs in the samples infected with the strains of non-Russian origin were associated with an increased risk of mortality (OR = 2.267, 95% confi dence interval 0.1594-8.653) compared to the SNPs in the samples obtained from the group of patients infected with the strains of Russian origin. Positive correlations were identifi ed between the average SNP number, nonsynonymous SNPs, and S-protein SNPs with the degree of respiratory failure, total NEWS score, CT-based form of disease, duration of treatment with mechanical ventilation, disease outcome, levels of LDH, glucose, D-dimer, and ferritin, and RNA amount in the PCR test. S-protein SNPs negatively correlated with the leukocyte and neutrophil counts.

Transcriptomic Analysis of Camellia Sinensis (L.) Kuntze Var. Niaowangensis Q. H. Chen Leaves Under Cold Stress

Fine varieties of the Yunwu Tribute Tea (Camellia Sinensis (L.) Kuntze var. niaowangensis Q. H. Chen) are distributed on the Yunwu Mountain, Guiding County, Guizhou province, China. Cold stress usually occurs in winter and is one of the most significant environmental factors restricting the growth of this plant as well as its geographical distribution. However, only few systematic studies have examined the molecular mechanism of cold tolerance in the Yunwu Tribute Tea. Hence, in this study, Illumina HiSeq technology was applied to investigate the cold-tolerance mechanism and for this purpose, cDNA libraries were obtained from two groups of samples namely, the cold-treated group (DW) and the control group (CK). A total of 185,973 unigenes were produced from 511,987 assembled transcripts and among these, 16,020 differentially expressed genes (DEGs) (corrected p-value <0.01, |log2(fold change)| >3), including 9,606 upregulated and 6,414 downregulated genes, were obtained. Moreover, the antioxidant enzyme system, plant hormone signal transduction, proline metabolism, tyrosine metabolism pathway, and transcription factors were analyzed and based on the results, a series of candidate genes related to cold stress were screened out and discussed. The physiological indexes related to the low temperature response were tested, along with five DEGs which were validated by quantitative real-time PCR. For this study, it is expected that the results of the transcriptome sequence of Yunwu Tribute Tea will provide valuable clues for genetic studies while helping to screen candidate genes for cold-resistance breeding in tea plants.

Physiological, Biochemical and Transcriptomic Analysis of the Aerial Parts (Leaf-Blade and Petiole) of Asarum sieboldii Responding to Drought Stress

Asarum sieboldii Miq. is a leading economic crop and a traditional medicinal herb in China. Leaf-blade and petiole are the only aerial tissues of A. sieboldii during the vegetative growth, playing a vital role in the accumulation and transportation of biomass energy. They also act as critical indicators of drought in agricultural management, especially for crops having underground stems. During drought, variations in the morphology and gene expression of the leaves and petioles are used to control agricultural irrigation and production. Besides, such stress can also alter the differential gene expression in these tissues. However, little is known about the drought-tolerant character of the aerial parts of A. sieboldii. In this study, we examined the physiological, biochemical and transcriptomic responses to the drought stress in the leaf blades and petioles of A. sieboldii. The molecular mechanism, involving in drought stress response, was elucidated by constructing the cDNA libraries and performing transcriptomic sequencing. Under drought stress, a total of 2,912 and 2,887 unigenes were differentially expressed in the leaf blade and petiole, respectively. The detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in drought tolerance. In response to drought, the leaf blade and petiole displayed a general physiological character, a higher SOD and POD activity, a higher MDA content and lower chlorophyll content. Three unigenes encoding POD were up-regulated, which can improve POD activity. Essential oil in petiole was extracted. The relative contents of methyleugenol and safrole in essential oil were increased from 0.01% to 0.05%, and 3.89% to 16.97%, respectively, while myristicin slightly reduced from 24.87% to 21.52%. Additionally, an IGS unigene, involved in eugenol biobiosynthesis, was found up-regulated under drought stress, which was predicated to be responsible for the accumulation of methyleugenol and safrole. Simple sequence repeats (SSRs) were characterized in of A. sieboldii, and a total of 5,466 SSRs were identified. Among them, mono-nucleotides were the most abundant repeat units, accounting for 44.09% followed by tri-, tetra-, penta and hexa-nucleotide repeats. Overall, the present work provides a valuable resource for the population genetics studies of A. sieboldii. Besides, it provides much genomic information for the functional dissection of the drought-resistance in A. sieboldii., which will be useful to understand the bio-regulatory mechanisms linked with drought-tolerance to enhance its yield.

Comparative transcriptomic analysis reveals key components controlling spathe color in Anthurium andraeanum (Hort.)

Anthurium andraeanum (Hort.) is an important ornamental in the tropical cut-flower industry. However, there is currently insufficient information to establish a clear connection between the genetic model(s) proposed and the putative genes involved in the differentiation between colors. In this study, 18 cDNA libraries related to the spathe color and developmental stages of A. andraeanum were characterized by transcriptome sequencing (RNA-seq). For the de novo transcriptome, a total of 114,334,082 primary sequence reads were obtained from the Illumina sequencer and were assembled into 151,652 unigenes. Approximately 58,476 transcripts were generated and used for comparative transcriptome analysis between three cultivars that differ in spathe color (‘Sasha’ (white), ‘Honduras’ (red), and ‘Rapido’ (purple)). A large number of differentially expressed genes (8,324), potentially involved in multiple biological and metabolic pathways, were identified, including genes in the flavonoid and anthocyanin biosynthetic pathways. Our results showed that the chalcone isomerase (CHI) gene presented the strongest evidence for an association with differences in color and the highest correlation with other key genes (flavanone 3-hydroxylase (F3H), flavonoid 3’5’ hydroxylase (F3’5’H)/ flavonoid 3’-hydroxylase (F3’H), and leucoanthocyanidin dioxygenase (LDOX)) in the anthocyanin pathway. We also identified a differentially expressed cytochrome P450 gene in the late developmental stage of the purple spathe that appeared to determine the difference between the red- and purple-colored spathes. Furthermore, transcription factors related to putative MYB-domain protein that may control anthocyanin pathway were identified through a weighted gene co-expression network analysis (WGCNA). The results provided basic sequence information for future research on spathe color, which have important implications for this ornamental breeding strategies.

De Novo Transcriptome Assembly for Venom Gland in Two Spe-Cies of Spiders (Sinopoda Pengi and Trichonephila Clavata)

Natural molecules from spider venom are considered potential drugs for diseases including cancer and pain, as well as the development of new biological insecticides for agricultural use. During coevolution in the long-term predator-prey game, spiders have formed a huge molecular diversity of toxins. As of March 1 of 2021, a total of 49,243 spider species had been described, but studies of venom have been performed in only a few hundred of these species due to the difficulty of collecting venom. Two technologies have helped partially dealing with this limitation in the recent past: the screening of cDNA libraries constructed from venom gland mRNAs and the heterologous expression of the coded peptides for functional characterization. In this study, transcriptomic analysis was performed to describe the predicted toxins of Sinopoda pengi (hereafter S. pengi) and Trichonephila clavata (hereafter T. clavata). The Trinity assembly result in 163,418 transcripts, 114,127 unigene of S. pengi and 125,099 transcripts, 87,084 unigene of T. clavata. A total of 22 and 24 unigenes were identified which were predicted to inhibitor cysteine knot (ICK) toxins from S. pengi and T. clavata, respectively. In summary, molecular templates with potential application value in medical and biological fields were obtained by classifying and characterizing presumed venom components, which lays a foundation for the further study of venom.

Transcriptome Analysis of Jujube (Ziziphus jujuba Mill.) Response to Heat Stress

Heat stress (HS) is a common stress influencing the growth and reproduction of plant species. Jujube (Ziziphus jujuba Mill.) is an economically important tree with strong abiotic stress resistance, but the molecular mechanism of its response to HS remains elusive. In this study, we subjected seedlings of Z. jujuba cultivar “Hqing1-HR” to HS (45°C) for 0, 1, 3, 5, and 7 days, respectively, and collected the leaf samples (HR0, HR1, HR3, HR5, and HR7) accordingly. Fifteen cDNA libraries from leaves were constructed for transcriptomics assays. RNA sequencing and transcriptomics identified 1,642, 4,080, 5,160, and 2,119 differentially expressed genes (DEGs) in comparisons of HR1 vs. HR0, HR3 vs. HR0, HR5 vs. HR0, and HR7 vs. HR0, respectively. Gene ontology analyses of the DEGs from these comparisons revealed enrichment in a series of biological processes involved in stress responses, photosynthesis, and metabolism, suggesting that lowering or upregulating expression of these genes might play important roles in the response to HS. This study contributed to our understanding of the molecular mechanism of jujube response to HS and will be beneficial for developing jujube cultivars with improved heat resistance.

CStone: A de novo transcriptome assembler for short-read data that identifies non-chimeric contigs based on underlying graph structure

With the exponential growth of sequence information stored over the last decade, including that of de novo assembled contigs from RNA-Seq experiments, quantification of chimeric sequences has become essential when assembling read data. In transcriptomics, de novo assembled chimeras can closely resemble underlying transcripts, but patterns such as those seen between co-evolving sites, or mapped read counts, become obscured. We have created a de Bruijn based de novo assembler for RNA-Seq data that utilizes a classification system to describe the complexity of underlying graphs from which contigs are created. Each contig is labelled with one of three levels, indicating whether or not ambiguous paths exist. A by-product of this is information on the range of complexity of the underlying gene families present. As a demonstration of CStones ability to assemble high-quality contigs, and to label them in this manner, both simulated and real data were used. For simulated data, ten million read pairs were generated from cDNA libraries representing four species, Drosophila melanogaster, Panthera pardus, Rattus norvegicus and Serinus canaria. These were assembled using CStone, Trinity and rnaSPAdes; the latter two being high-quality, well established, de novo assembers. For real data, two RNA-Seq datasets, each consisting of ≈30 million read pairs, representing two adult D. melanogaster whole-body samples were used. The contigs that CStone produced were comparable in quality to those of Trinity and rnaSPAdes in terms of length, sequence identity of aligned regions and the range of cDNA transcripts represented, whilst providing additional information on chimerism. Here we describe the details of CStones assembly and classification process, and propose that similar classification systems can be incorporated into other de novo assembly tools. Within a related side study, we explore the effects that chimera’s within reference sets have on the identification of differentially expression genes. CStone is available at: https://sourceforge.net/projects/cstone/.

Transcriptomics and Metabolomics Changes Triggered by Inflorescence Removal in Panax notoginseng (Burk.)

The root of Panax notoginseng (Burk.), in which saponins are the major active components, is a famous traditional Chinese medicine used to stop bleeding and to decrease inflammation and heart disease. Inflorescence removal increases the yield and quality of P. notoginseng, but the underlying molecular mechanisms are unknown. Here, the differences between inflorescence-removal treatment and control groups of P. notoginseng were compared using transcriptomics and metabolomics analyses. Illumina sequencing of cDNA libraries prepared from the rhizomes, leaves and roots of the two groups independently identified 6,464, 4,584, and 7,220 differentially expressed genes (DEG), respectively. In total, 345 differentially expressed transcription factors (TFs), including MYB and WRKY family members, were induced by the inflorescence-removal treatment. Additionally, 215 DEGs involved in saponin terpenoid backbone biosynthetic pathways were identified. Most genes involved in the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways were activated by inflorescence removal. The co-expression analysis showed that the low expression levels of flavonoid biosynthesis-related genes (e.g., C4H and F3H) decreased the biosynthesis and accumulation of some flavonoids after inflorescence removal. The results not only provide new insights into the fundamental mechanisms underlying the poorly studied inflorescence-removal process in P. notoginseng and other rhizome crops, but they also represent an important resource for future research on gene functions during inflorescence-removal treatments and the reproductive stage.