- Background: Long non-coding RNAs (lncRNAs) are important component of mammalian genomes, where their numbers are even larger than that of protein-coding genes. - 22,630) have more lncRNA genes than protein-coding genes in the. - Recently, mammalian lncRNAs were reported to play critical roles in immune response to influenza A virus infections. - We then collected 151,970 assembled transcripts from RNA-Seq data of 21 individuals from three tissues and annotated 4094 duck lncRNAs. - Comparing to duck protein-coding transcripts, we found that 4094 lncRNAs had smaller number of exons (2.4 vs. - These data and analysis provide information for duck lncRNAs ’ function in immune response to influenza A virus.. - found that some lncRNAs (such as DWORF [5] and SPAR [6]) encoded peptides and played important roles in the myocardial contraction and muscle regeneration, respectively. - Duck is one of the important economical waterfowl and the natural reservoir of all influenza A viruses har- boring 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes [10], with the exception of the H13, H16, H17 and H18 subtypes [11]. - Full list of author information is available at the end of the article. - Here, we developed a systematical pipeline for lncRNA identification and annotated 4094 duck lncRNAs using brain, lung and spleen transcriptomes of control individ- uals and ones that were infected with a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/. - Hubei/65/05) H5N1 virus (DK/49 or GS/65). - We com- pared the genomic structure and expression pattern of duck lncRNAs to their protein-coding genes and identi- fied the differentially expressed lncRNAs in H5N1 virus infected duck and control individuals. - We further identi- fied lncRNAs and protein-coding genes co-expressional modules. - Genome-wide annotation of duck lncRNAs. - Hubei/65/05) H5N1 virus after 1, 2 and 3 days were as- sembled to produce 151,970 transcripts (Additional file 2:. - 1 to identify duck lncRNAs. - 1 Schematic diagram of the pipeline for identification of duck lncRNAs. - Putative lncRNAs were identified using five criteria: (1) filtered with duck protein-coding genes. - We further filtered the remained 39,076 transcripts and removed 3731 transcripts being homologous to protein-coding genes in the three protein databases (Swiss-Prot, KEGG and NR). - After that, we assessed the protein-coding potential of 35,345 transcripts using the CPC program and deleted 36 potential coding tran- scripts [21]. - 1 and Additional file 3). - In order to iden- tify the accuracy and quality of these annotated duck lncRNAs, we blasted them to the non-redundant data- base in the NCBI (RefSeq) and found 752 were anno- tated in this database.. - locations relative to the nearest protein-coding genes.. - Among these 4094 duck lncRNAs, a large proportion (87.6%) was located in the intergenic regions, 8.4% was antisense transcripts of protein-coding genes, while only 4.0% was overlapped lncRNAs (Fig. - We further counted the distribution of transcript length, exon num- ber and expression level of 4094 lncRNAs and compared them to 16,353 duck protein-coding transcripts. - In contrast, the average length of duck mRNAs (1687 nt) was smaller than that of duck lncRNAs. - For gene structure, duck lncRNAs contained two to nine exons with an aver- age of 2.4 exons. - We further compared expressional patterns of lncRNAs and protein-coding genes in three tissues (brain, lung and spleen). - The average expression levels of the pu- tative lncRNA genes (brain average 15.7 FPKM, lung average 10.7 FPKM and spleen average 9.9 FPKM) tend to be lower than those of the protein-coding. - 2 Categories and features of the 4094 predicted duck lncRNAs. - a Categories of duck lncRNAs divided based on genomic location between lncRNAs and protein-coding genes. - strand for each of the three main types was labeled on the columns. - b-d Duck lncRNAs are longer, have fewer exons and have lower expression levels than protein-coding genes. - Expression (b), distribution of length (c) and number of exons (d) of 4094 lncRNAs and 16,353 protein-coding transcripts of duck (BGI_duck_1.0). - Identification of differentially expressed duck lncRNAs (DElncRNAs). - We compared lncRNA expression in duck infected with H5N1 viruses (DK/49-infected or GS/65-infected) against control individuals and identified a total of 619 DElncRNAs (FDR ≤ 0.05, fold change ≥ 2) including 323, 217 and 206 DElncRNAs in the brain, lung and spleen respectively (Fig. - 3a and Additional file 4: Table S2). - 3a and Additional file 4:. - These results suggested that the DElncRNAs might be involved into immune response to influenza A virus infection in ducks.. - Prediction the target protein-coding gene of lncRNA in cis and in trans. - These lncRNAs function in cis, acting on linked genes in the vicinity of the RNA’s site. - In order to identify duck lncRNAs functioning in cis, we predicted the potential protein-coding gene targets of lncRNAs among differen- tially expressed genes (DEGs) using 10 kb and 100 kb as the cutoff (Table 1). - We detected lncRNAs-coding genes pairs when used the 10/100 kb as the cutoff, and 721/106 DElncRNAs-DEGs pairs when used the 10/100 kb as the cutoff. - The lncRNAs-coding genes co-expression networks were performed with 619 DElncRNAs and 5594 DEGs using. - As a consequence, 13 co-expression modules were con- structed in size from 2261 genes in the turquoise mod- ule to 36 genes in the salmon module (Fig. - 4a and Additional file 8: Table S6).. - Functional analysis indicated that 5594 expressed DEGs were enriched in 640 GO terms (401 GO under biological process, 144 GO under cellular component and 95 GO under molecular function) (Additional file 9:. - The results showed that DEGs from magenta and green modules were associated with immune re- sponse in the biological process, including innate im- mune response (GO defense response to virus (GO positive regulation of defense re- sponse to virus by host (GO immune re- sponse (GO: 0006955) and negative regulation of viral genome replication (GO: 0045071).. - 5), supporting their critical role in host response to influenza A virus infection. - Such inference was further supported by pathway analysis (KEGG), which demon- strated that DEGs in the magenta and green modules were significantly enriched (p <. - lncRNAs Coding genes DElncRNAs DEGs lncRNA-coding gene pairs DElncRNA-DEGs pairs. - We searched for protein-coding genes 100 kb/10 kb upstream and downstream of the identified lncRNAs. - Orange nodes represent lncRNAs, green nodes represent coding genes. - The line between lncRNAs and coding genes indicates an expression pattern correlation. - hubei H5N1 virus after 36 h. - In the present study, we built a screening platform to identify lncRNAs after explored features of 62,447 lncRNAs from five model animals (Fig. - This pipeline removed known protein-coding genes using the most effective computational methods available to date, such as Cufflinks and CPC. - Unlike the previously reserved intergenic transcripts [31], we retained the intergenic transcripts, antisense transcripts and transcripts that overlap with the protein-coding genes. - In addition, we filtered the potential protein-coding genes through blasting with duck protein sequences, chicken protein sequences and other. - Interestingly, TRIM25, IFNK, DDX58 (RIG-I), AvIFIT and OASL were found to be a highly connected gene in the magenta module, and IL8, FADD, NF- κ B, IRF7, IFNE, IFNA and CXCL6 in the green module, these protein-coding genes play import- ant roles in anti-virus immune response (Fig. - [33] found an IFN-induced long noncoding RNA lnc-Lsm3b, played a negative feedback regulatory role in the late immune response and. - 0.2) in the magenta and green modules. - These three lncRNAs showed differentially expressed in all three H5N1 infections in vivo and in vitro, implying that might affect function of RIG-I in the immune response influenza A viruses and be interested to further functional studies.. - These include: (1) some duck lncRNAs lacking polyadenylation might be not annotated using mRNA transcriptome. - (2) due to the inherent limitations of using 90 bp paired-end RNA-Seq, we did not get full se- quences of all annotated duck lncRNAs. - (3) some lncRNAs may be annotated incorrectly due to error of the available duck assembly.. - We first identified duck lncRNAs associated with immune response to avian influenza virus using RNA-Seq data. - We present a pipeline to investigate the duck lncRNAs and predicted the function of lncRNAs based on the coding gene neighbor loci and the enriched functions of co-expression protein-coding genes. - These analyses together with duck lncRNA sequences provide information to understand their functions in the im- mune response.. - After clean reads, we build the index of the reference genome [14] using Bowtie v2.2.6 and aligned paired-end clean reads to the reference genome using Tophat v2.1.0. - Describing feature of lncRNAs and protein-coding genes A total of 4094 lncRNAs and 16,353 mRNAs were char- acterized in transcript length, exon number and expres- sional profiles. - The proportion of different types of lncRNAs and protein-coding transcripts were calculated. - Furthermore, we used brain, lung and spleen transcriptomes of control ducks to characterize the expression pattern of the lncRNA genes and coding genes, whose FPKM value were calculated using the Cufflinks (v2.2.1) program.. - Protein-coding genes were within 10 kb/100 kb upstream and downstream of the iden- tified lncRNAs were inferred as neighbor target genes.. - After that, we constructed networks using the blockwiseModules function in the WGCNA package with the minimum module size to 30 genes, and the minimum height for merging modules at 0.15 (default value). - with H5N1 virus. - PCR primers are listed in Additional file 11: Table S9.. - Additional file 1: Figure S1. - Length distribution of duck lncRNAs transcript (A), ORF length (B) and numbers of exons (C) were shown in the picture. - (TIF 3391 kb) Additional file 2: Table S1. - Additional file 3: Information of lncRNA annotated with GTF files.. - Additional file 4: Table S2. - Additional file 5: Table S3. - Protein-coding genes located within 10/. - 100 kb upstream and downstream of duck lncRNAs. - (XLSX 642 kb) Additional file 6: Table S4. - Additional file 7: Table S5. - Additional file 8: Table S6. - Additional file 9: Table S7. - Additional file 10: Table S8. - Additional file 11: Table S9. - DK/49: A highly pathogenic (A/duck/Hubei/49/05) H5N1 virus;. - GS/65: A weakly pathogenic (A/goose/Hubei/65/05) H5N1 virus. - Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. - Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. - Identification of long non- protein-coding RNAs in chicken skeletal muscle using next generation sequencing. - Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. - CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. - Exploring the function of long non-coding RNA in the development of bovine early embryos. - Genome-Wide Analysis of lncRNA and mRNA Expression During Differentiation of Abdominal Preadipocytes in the Chicken
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