De novo identification of viral pathogens from cell culture hologenomes

<p>Abstract</p> <p>Background</p> <p>Fast, specific identification and surveillance of pathogens is the cornerstone of any outbreak response system, especially in the case of emerging infectious diseases and viral epidemics. This process is generally tedious and time-consuming thus making it ineffective in traditional settings. The added complexity in these situations is the non-availability of pure isolates of pathogens as they are present as mixed genomes or hologenomes. Next-generation sequencing approaches offer an attractive solution in this scenario as it provides adequate depth of sequencing at fast and affordable costs, apart from making it possible to decipher complex interactions between genomes at a scale that was not possible before. The widespread application of next-generation sequencing in this field has been limited by the non-availability of an efficient computational pipeline to systematically analyze data to delineate pathogen genomes from mixed population of genomes or hologenomes.</p> <p>Findings</p> <p>We applied next-generation sequencing on a sample containing mixed population of genomes from an epidemic with appropriate processing and enrichment. The data was analyzed using an extensive computational pipeline involving mapping to reference genome sets and <it>de-novo </it>assembly. In depth analysis of the data generated revealed the presence of sequences corresponding to <it>Japanese encephalitis </it>virus. The genome of the virus was also independently <it>de-novo </it>assembled. The presence of the virus was in addition, verified using standard molecular biology techniques.</p> <p>Conclusions</p> <p>Our approach can accurately identify causative pathogens from cell culture hologenome samples containing mixed population of genomes and in principle can be applied to patient hologenome samples without any background information. This methodology could be widely applied to identify and isolate pathogen genomes and understand their genomic variability during outbreaks.</p

Dynamic Expression of Long Non-Coding RNAs (lncRNAs) in Adult Zebrafish

<div><p>Long non-coding RNAs (lncRNA) represent an assorted class of transcripts having little or no protein coding capacity and have recently gained importance for their function as regulators of gene expression. Molecular studies on lncRNA have uncovered multifaceted interactions with protein coding genes. It has been suggested that lncRNAs are an additional layer of regulatory switches involved in gene regulation during development and disease. LncRNAs expressing in specific tissues or cell types during adult stages can have potential roles in form, function, maintenance and repair of tissues and organs. We used RNA sequencing followed by computational analysis to identify tissue restricted lncRNA transcript signatures from five different tissues of adult zebrafish. The present study reports 442 predicted lncRNA transcripts from adult zebrafish tissues out of which 419 were novel lncRNA transcripts. Of these, 77 lncRNAs show predominant tissue restricted expression across the five major tissues investigated. Adult zebrafish brain expressed the largest number of tissue restricted lncRNA transcripts followed by cardiovascular tissue. We also validated the tissue restricted expression of a subset of lncRNAs using independent methods. Our data constitute a useful genomic resource towards understanding the expression of lncRNAs in various tissues in adult zebrafish. Our study is thus a starting point and opens a way towards discovering new molecular interactions of gene expression within the specific adult tissues in the context of maintenance of organ form and function.</p></div

RNA-sequencing data production and alignment results for tissue-specific Poly (A) reads.

<p>The total number of sequence reads obtained from the five zebrafish tissues using RNA sequencing is described. Mapped reads represent all transcripts that aligned back to the zebrafish reference genome (Zv9).</p

Real time assay for putative tissue restricted lncRNAs.

<p>Expression of candidate lncRNA transcripts was analyzed by semi quantitative RT-PCR in A) heart; B) liver; C) muscle; D) brain and E) blood tissues. A tissue specific protein coding marker gene viz <i>cmlc2</i> (heart); <i>tfr</i> (liver); <i>mdka</i> (brain); <i>murcb</i> (muscle) and <i>tal1</i> (blood) was used as standard control. See text for details on selection of protein coding marker genes. LncRNA transcripts investigated for a particular tissue type showed relatively predominant expression in the specific tissue when compared with other tissues.</p

Distribution of embryonic lncRNA transcripts in adult tissues of zebrafish.

<p>A. Clustered heat maps of 2,266 lncRNA transcripts obtained from Pauli et(H), liver (L), muscle (M), brain (Br) and blood (Bl) are represented. The color key represents the FPKM values in which grey color indicates the range from 0 to 10, light blue indicates the range from 11 to 100 and dark blue indicates 101 and above FPKM values for those with the highest expression. B. Enlarged section of the heat map depicting differential expression profile of 90 lncRNA transcripts expression across five tissues.</p

LncRNAs show tissue restricted expression patterns.

<p>Whole mount <i>in situ</i> hybridization of lncRNA transcripts. Shown are images with probes specific to the two indicated brain restricted lncRNAs. Arrow heads indicate the expression domains. A and B Anatomical cartoons of 24 hpf developing zebrafish embryo and adult zebrafish brain. C and D Expression of lncRNA transcript <i>lncBrHM_035.</i> (C) Dorsal view (anterior up) and lateral view (anterior to the left) showing expression in mid-hind brain boundary and hind brain of 24hpf zebrafish embryos. (D) Dorsal view (anterior up) of the adult zebrafish brain showing expression in regions of cerebellar crest (CC). E and F Expression of lncRNA transcript <i>lncBrM_002.</i> (E) Dorsal view (anterior up) and lateral view (anterior to the left) showing expression in fore-brain (FB), mid-hind brain boundary (MHB) and hind brain (HB) of 24hpf zebrafish embryos. (F) Dorsal view (anterior up) of the adult zebrafish brain showing expression in the regions of CC and a localized signal in eminentia granularis (EG). MB, mid brain; OB, olfactory bulb; Tel, telencephalon; Ha, habenula; Teo, optic tectum; MO, medulla oblongata.</p