Search

Abstract Background Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. Methods The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2–4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. Results The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. Conclusions The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.

Abstract Background Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. Methods Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. Results Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. Conclusions Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Abstract Background There are a large number of biological databases publicly available for scientists in the web. Also, there are many private databases generated in the course of research projects. These databases are in a wide variety of formats. Web standards have evolved in the recent times and semantic web technologies are now available to interconnect diverse and heterogeneous sources of data. Therefore, integration and querying of biological databases can be facilitated by techniques used in semantic web. Heterogeneous databases can be converted into Resource Description Format (RDF) and queried using SPARQL language. Searching for exact queries in these databases is trivial. However, exploratory searches need customized solutions, especially when multiple databases are involved. This process is cumbersome and time consuming for those without a sufficient background in computer science. In this context, a search engine facilitating exploratory searches of databases would be of great help to the scientific community. Results We present BioCarian, an efficient and user-friendly search engine for performing exploratory searches on biological databases. The search engine is an interface for SPARQL queries over RDF databases. We note that many of the databases can be converted to tabular form. We first convert the tabular databases to RDF. The search engine provides a graphical interface based on facets to explore the converted databases. The facet interface is more advanced than conventional facets. It allows complex queries to be constructed, and have additional features like ranking of facet values based on several criteria, visually indicating the relevance of a facet value and presenting the most important facet values when a large number of choices are available. For the advanced users, SPARQL queries can be run directly on the databases. Using this feature, users will be able to incorporate federated searches of SPARQL endpoints. We used the search engine to do an exploratory search on previously published viral integration data and were able to deduce the main conclusions of the original publication. BioCarian is accessible via http://www.biocarian.com . Conclusions We have developed a search engine to explore RDF databases that can be used by both novice and advanced users.