Baiting out a full length sequence from unmapped RNA-seq data

Background As a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored. Results We develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure. Conclusion We believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.

Complete Genome Analysis of a Novel Picorna-Like Virus From a Ladybird Beetle, Cheilomenes Sexmaculata

The ladybird beetle Cheilomenes sexmaculata (family Coccinellidae, order Coleoptera), is a common insect predator of agricultural pests. In this study, the full genome sequence of a novel picorna-like virus, temporarily named “Cheilomenes sexmaculata picorna-like virus 1” (CSPLV1), was identified from C. sexmaculata. The full-length sequence of CSPLV1 was 11,384 nucleotide (nt) in length (excluding the polyA tail) with one predicted open reading frame (ORF) encoding 3727 amino acids, a 13 nt 5' untranslated region (UTR) and a 187 nt 3' UTR. The ORF of CSPLV1 consisted of four distinct domains including an RNA virus helicase domain (3029-3319 nt), a peptidase domain (5555-6121 nt), an RNA-dependent RNA polymerase domain (7154-8101 nt) and a picorna-like coat protein domain (8606-9283nt). Phylogenetic analysis based on the conserved RdRP sequence showed that CSPLV1, together with Wuhan house centipede virus 3, Hypera postica associated virus 1 and Diabrotica undecimpunctata virus 1, formed as an unclassified group which is closely related to the clade Solinviviridae. To the best of our knowledge, CSPLV1 is the first picorna-like virus revealed in C. sexmaculata.

LncRNA NR038975, A Serum-Based Biomarker, Promotes Gastric Tumorigenesis by Interacting With NF90/NF45 Complex

Gastric cancer (GC) is one of the deadliest cancers, and long noncoding RNAs (lncRNAs) have been reported to be the important regulators during the occurrence and development of GC. The present study identified a novel and functional lncRNA in GC, named NR038975, which was confirmed to be markedly upregulated in the Gene Expression Profiling Interactive Analysis (GEPIA) dataset and our independent cohort of GC tissues. We firstly characterized the full-length sequence and subcellular location of NR038975 in GC cells. Our data demonstrated that upregulated NR038975 expression was significantly related to lymph node metastasis and TNM stage. In addition, knockdown of NR038975 inhibited GC cell proliferation, migration, invasion, and clonogenicity and vice versa. Mechanistically, RNA pull-down and mass spectrometry assays identified the NR038975-binding proteins and NF90/NF45 complex, and the binding was also confirmed by RNA immunoprecipitation and confocal experiments. We further demonstrated that genetic deficiency of NR038975 abrogated the interaction between NF45 and NF90. Moreover, NF90 increased the stability of NR038975. Thus, NR038975-NF90/NF45 will be an important combinational target of GC. Finally, we detected NR038975 in serum exosomes and serum of GC patients. Our results indicated that NR038975 was a biomarker for gastric tumorigenesis. The current study demonstrated that NR038975 is a novel lncRNA that is clinically and functionally engaged in GC progression and might be a novel diagnostic marker and potential therapeutic target.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Identification, Expression, and Functional Characterization of ScCaM in Response to Various Stresses in Sugarcane

Calmodulin (CaM), as an important factor in the calcium signaling pathway, is widely involved in plant growth and development regulation and responses to external stimuli. In this study, the full-length sequence of the ScCaM gene (GenBank: GQ246454) was isolated from the leaves of a Saccharum spp. hybrid. Prokaryotic expression showed that ScCaM could be solubly expressed and purified in Escherichia coli BL21. Subcellular localization confirmed that ScCaM was localized in the plasma membrane and nucleus of cells. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that ScCaM can be induced by various stresses, including sodium chloride (NaCl), chromium trichloride (CrCl3), salicylic acid (SA), and methyl jasmonate (MeJA). Ectopic expression in Arabidopsis thaliana demonstrated that ScCaM can affect the growth and development of transgenic plants. Moreover, the qRT-PCR analysis indicated that the overexpression of the allogenic ScCaM gene inhibits the expression of AtSTM, leading to the phenomenon of multiple-tillering in transgenic A. thaliana. Furthermore, the expression patterns of ScCaM under abiotic stress and phytohormone stimulation in transgenic A. thaliana confirmed that ScCaM was involved in the responses to phytohormone, high salt, and heavy metal stresses. The present study provided valuable information and facilitates further investigation into the function of ScCaM in the future.

A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

A Simple and Fast Method to Sequence the Full-Length Spike Gene for SARS-CoV-2 Variant Identification from Patient Samples

Since the beginning of the pandemic, a race has been underway to detect SARS-CoV-2 virus infection (PCR screening, serological diagnostic kits), treat patients (drug repurposing, standard care) and develop a vaccine. After almost a year of active circulation worldwide, SARS-CoV-2 variants have appeared in different countries. Those variants include mutations in multiple regions of the genome, particularly in the spike gene. Because this surface protein is a key player in both the spread of the virus and the efficacy of vaccine strategies, the challenge is to efficiently monitor the appearance of spike mutations in the population. The present work describes a procedure based on the widely available Sanger technology to produce a full-length sequence of the spike gene from patient-derived samples.