Discovery of EST-SSRs in Lung Cancer: Tagged ESTs with SSRs Lead to Differential Amino Acid and Protein Expression Patterns in Cancerous Tissues

Tandem repeats are found in both coding and non-coding sequences of higher organisms. These sequences can be used in cancer genetics and diagnosis to unravel the genetic basis of tumor formation and progression. In this study, a possible relationship between SSR distributions and lung cancer was studied by comparative analysis of EST-SSRs in normal and lung cancerous tissues. While the EST-SSR distribution was similar between tumorous tissues, this distribution was different between normal and tumorous tissues. Trinucleotides tandem repeats were highly different; the number of trinucleotides in ESTs of lung cancer was 3 times higher than normal tissue. Significant negative correlation between normal and cancerous tissue showed that cancerous tissue generates different types of trinucleotides. GGC and CGC were the more frequent expressed trinucleotides in cancerous tissue, but these SSRs were not expressed in normal tissue. Similar to the EST level, the expression pattern of EST-SSRs-derived amino acids was significantly different between normal and cancerous tissues. Arg, Pro, Ser, Gly, and Lys were the most abundant amino acids in cancerous tissues, and Leu, Cys, Phe, and His were significantly more abundant in normal tissues than in cancerous tissues. Next, the putative functions of triplet SSR-containing genes were analyzed. In cancerous tissue, EST-SSRs produce different types of proteins. Chromodomain helicase DNA binding proteins were one of the major protein products of EST-SSRs in the cancerous library, while these proteins were not produced from EST-SSRs in normal tissue. For the first time, the findings of this study confirmed that EST-SSRs in normal lung tissues are different than in unhealthy tissues, and tagged ESTs with SSRs cause remarkable differences in amino acid and protein expression patterns in cancerous tissue. We suggest that EST-SSRs and EST-SSRs differentially expressed in cancerous tissue may be suitable candidate markers for lung cancer diagnosis and prediction

Deciphering the role of alternative splicing as a potential regulator in fat-tail development of sheep: a comprehensive RNA-seq based study

Abstract Although research on alternative splicing (AS) has been widely conducted in mammals, no study has investigated the splicing profiles of genes involved in fat-tail formation in sheep. Here, for the first time, a comprehensive study was designed to investigate the profile of AS events and their involvement in fat-tail development of sheep. In total, 45 RNA-Seq samples related to seven different studies, which have compared the fat-tailed vs thin-tailed sheep breeds, were analyzed. Two independent tools, rMATS and Whippet, along with a set of stringent filters were applied to identify differential AS (DAS) events between the breeds per each study. Only DAS events that were detected by both tools as well as in at least three datasets with the same ΔPSI trend (percent spliced in), were considered as the final high-confidence set of DAS genes. Final results revealed 130 DAS skipped exon events (69 negative and 61 positive ΔPSI) belonged to 124 genes. Functional enrichment analysis highlighted the importance of the genes in the underlying molecular mechanisms of fat metabolism. Moreover, protein–protein interaction network analysis revealed that DAS genes are significantly connected. Of DAS genes, five transcription factors were found that were enriched in the biological process associated with lipid metabolism like “Fat Cell Differentiation”. Further investigations of the findings along with a comprehensive literature review provided a reliable list of candidate genes that may potentially contribute to fat-tail formation including HSD11B1, SIRT2, STRN3 and TCF7L2. Based on the results, it can be stated that the AS patterns may have evolved, during the evolution of sheep breeds, as another layer of regulation to contribute to biological complexity by reprogramming the gene regulatory networks. This study provided the theoretical basis of the molecular mechanisms behind the sheep fat-tail development in terms of AS

RNA-Seq based genetic variant discovery provides new insights into controlling fat deposition in the tail of sheep

Abstract Genetic basis of fat deposition in sheep tail have not been completely elucidated yet. Understanding the genetic mechanisms controlling fat-tail size can improve breeding strategies to modulate fat deposition. RNA sequencing has made it possible to discover genetic variants that may underlie various phenotypic differences. Hence, to identify genetic variants that are important for describing different fat-tail phenotypes in sheep, RNA sequencing was used for single nucleotide polymorphism (SNP) calling in two Iranian sheep breeds (Lori-Bakhtiari, fat-tailed; n = 4, vs Zel, thin-tailed; n = 4). Using a stringent pipeline, a total of 112,344 known SNPs were genotyped, of which 30,550 and 42,906 SNPs were shared by at least two Lori-Bakhtiari and Zel, respectively. Comparing these SNPs showed 2,774 (including 209 missense and 25 deleterious SNPs) and 10,470 (including 1,054 missense and 116 deleterious SNPs) breed-specific SNPs in Lori-Bakhtiari and Zel sheep, respectively. Potential breed-specific SNPs were detected by considering those located in QTL regions associated with fatness or reported as important candidates in previous similar studies. Of the breed-specific SNPs, 724 and 2,905 were located in the QTL regions. Functional enrichment analysis of the affected genes revealed several enriched gene ontologies and KEGG pathways related to fat metabolism. Based on the results, several affected genes were proposed to be strongly linked with fat deposition such as DGAT2, ACSL1, ACACA, ADIPOQ, ACLY, FASN, CPT2, SCD, ADCY6, PER3, CSF1R, SLC22A4, GFPT1, CDS2, BMP6, ACSS2, ELOVL6, HOXA10 and FABP4. Moreover, several SNPs were found in the candidate genes related to fatty acid oxidation introducing them as promising candidates responsible for lower fat content in tail of Zel. Our findings provided new insights into the genetic mechanisms of fat deposition in sheep, which can serve to designing appropriate breeding programs

Identification and Expression Analysis of Long Noncoding RNAs in Fat-Tail of Sheep Breeds

Emerging evidence suggests that long non-coding RNAs (lncRNAs) participate in the regulation of a diverse range of biological processes. However, most studies have been focused on a few established model organisms and little is known about lncRNAs in fat-tail development in sheep. Here, the first profile of lncRNA in sheep fat-tail along with their possible roles in fat deposition were investigated, based on a comparative transcriptome analysis between fat-tailed (Lori-Bakhtiari) and thin-tailed (Zel) Iranian sheep breeds. Among all identified lncRNAs candidates, 358 and 66 transcripts were considered novel intergenic (lincRNAs) and novel intronic (ilncRNAs) corresponding to 302 and 58 gene loci, respectively. Our results indicated that a low percentage of the novel lncRNAs were conserved. Also, synteny analysis identified 168 novel lincRNAs with the same syntenic region in human, bovine and chicken. Only seven lncRNAs were identified as differentially expressed genes between fat and thin tailed breeds. Q-RT-PCR results were consistent with the RNA-Seq data and validated the findings. Target prediction analysis revealed that the novel lncRNAs may act in cis or trans and regulate the expression of genes that are involved in the lipid metabolism. A gene regulatory network including lncRNA-mRNA interactions were constructed and three significant modules were found, with genes relevant to lipid metabolism, insulin and calcium signaling pathway. Moreover, integrated analysis with AnimalQTLdb database further suggested six lincRNAs and one ilncRNAs as candidates of sheep fat-tail development. Our results highlighted the putative contributions of lncRNAs in regulating expression of genes associated with fat-tail development in sheep

Genome-Wide Identification and Analysis of Variants in Domestic and Wild Bactrian Camels Using Whole-Genome Sequencing Data

The population size of Bactrian camels is smaller than dromedary, and they are distributed in cold and mountain regions and are also at the risk of extinction in some countries such as Iran. To identify and investigate the genome-wide variations, whole-genome sequencing of two Iranian Bactrian camels were performed with 37.4- and 42.6-fold coverage for the first time. Along with Iranian Bactrian camels, sequencing data from two Mongolian domestic and two wild Bactrian camels deposited in the NCBI were reanalyzed. The analysis eventuated to the identification of 4,908,998, 4,485,725, and 4,706,654 SNPs for Iranian, Mongolian domestic, and wild Bactrian camels, respectively. Also, INDEL variations ranged from 358,311 to 533,188 in all six camels. Results of variants annotation in all samples revealed that more than 88 percent of SNPs and INDELs were located in the intergenic and intronic regions. We found that 800,530 SNPs were common among all studied camels, containing 4,046 missense variants that affected 2,428 genes. Investigation of common genes among all camels containing the missense SNPs showed that there are 98 zinc finger and 4 fertility-related genes (ZP1, ZP2, ZP4, and ZPBP) in this set

Genome-wide identification and analysis of A-to-I RNA editing events in bovine by transcriptome sequencing.

RNA editing increases the diversity of the transcriptome and proteome. Adenosine-to-inosine (A-to-I) editing is the predominant type of RNA editing in mammals and it is catalyzed by the adenosine deaminases acting on RNA (ADARs) family. Here, we used a largescale computational analysis of transcriptomic data from brain, heart, colon, lung, spleen, kidney, testes, skeletal muscle and liver, from three adult animals in order to identify RNA editing sites in bovine. We developed a computational pipeline and used a rigorous strategy to identify novel editing sites from RNA-Seq data in the absence of corresponding DNA sequence information. Our methods take into account sequencing errors, mapping bias, as well as biological replication to reduce the probability of obtaining a false-positive result. We conducted a detailed characterization of sequence and structural features related to novel candidate sites and found 1,600 novel canonical A-to-I editing sites in the nine bovine tissues analyzed. Results show that these sites 1) occur frequently in clusters and short interspersed nuclear elements (SINE) repeats, 2) have a preference for guanines depletion/enrichment in the flanking 5'/3' nucleotide, 3) occur less often in coding sequences than other regions of the genome, and 4) have low evolutionary conservation. Further, we found that a positive correlation exists between expression of ADAR family members and tissue-specific RNA editing. Most of the genes with predicted A-to-I editing in each tissue were significantly enriched in biological terms relevant to the function of the corresponding tissue. Lastly, the results highlight the importance of the RNA editome in nervous system regulation. The present study extends the list of RNA editing sites in bovine and provides pipelines that may be used to investigate the editome in other organisms

Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of <i>Capra hircus</i>

<div><p>Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, <i>Capra hircus</i>, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes.</p> </div

Genome-Wide Characterization of RNA Editing Sites in Primary Gastric Adenocarcinoma through RNA-seq Data Analysis

RNA editing is a posttranscriptional nucleotide modification in humans. Of the various types of RNA editing, the adenosine to inosine substitution is the most widespread in higher eukaryotes, which is mediated by the ADAR family enzymes. Inosine is recognized by the biological machinery as guanosine; therefore, editing could have substantial functional effects throughout the genome. RNA editing could contribute to cancer either by exclusive editing of tumor suppressor/promoting genes or by introducing transcriptomic diversity to promote cancer progression. Here, we provided a comprehensive overview of the RNA editing sites in gastric adenocarcinoma and highlighted some of their possible contributions to gastric cancer. RNA-seq data corresponding to 8 gastric adenocarcinoma and their paired nontumor counterparts were retrieved from the GEO database. After preprocessing and variant calling steps, a stringent filtering pipeline was employed to distinguish potential RNA editing sites from SNPs. The identified potential editing sites were annotated and compared with those in the DARNED database. Totally, 12362 high-confidence adenosine to inosine RNA editing sites were detected across all samples. Of these, 12105 and 257 were known and novel editing events, respectively. These editing sites were unevenly distributed across genomic regions, and nearly half of them were located in 3′UTR. Our results revealed that 4868 editing sites were common in both normal and cancer tissues. From the remaining sites, 3985 and 3509 were exclusive to normal and cancer tissues, respectively. Further analysis revealed a significant number of differentially edited events among these sites, which were located in protein coding genes and microRNAs. Given the distinct pattern of RNA editing in gastric adenocarcinoma and adjacent normal tissue, edited sites have the potential to serve as the diagnostic biomarkers and therapeutic targets in gastric cancer

Gene expression profile analysis of residual feed intake for Isfahan native chickens using RNA-SEQ data

The factors that affect residual feed intake (RFI) of two chicken breeds (Ross as commercial and Isfahan as Iranian local breed with low and high RFI, respectively) were examined. We sequenced their liver transcriptomes using RNA-Seq while focusing on the identification of important candidate genes, which might influence RFI and growth rate. The differential gene expression analysis revealed that 121 and 279 known genes were significantly up- and down-regulated in the local breed, respectively. QTL enrichment analysis revealed that 63 down-regulated genes in the local breed were enriched in the QTL regions related to feed efficiency traits. Moreover, the functional enrichment analysis showed that the down-regulated genes in the native chickens were mainly involved in the different metabolic processes, such as carboxylic acid metabolic process and response to stress. The Differentially Expressed Genes explored between the chickens of the two breeds led to the identification of some important candidate genes for further breed improvement programmes, including RSAD2, IL15, LIPI, EGR1 and DUSP16. These findings will be a useful resource for the biological investigations of RFI-related genes in Isfahan local chickens and may provide some clues for understanding the molecular genetic mechanisms in other chicken breeds.highlights A global view of transcriptome differences in the liver tissues of commercial and local chicken breeds with different RFI values is obtained. This investigation will contribute to the improvement of chicken genome annotation through the identification of novel transcripts and novel gene. Some important candidate genes related to RFI is obtained

Combining independent de novo assemblies to optimize leaf transcriptome of Persian walnut.

Transcriptome resources can facilitate to increase yield and quality of walnuts. Finding the best transcriptome assembly has not been the subject of walnuts research as yet. This research generated 240,179,782 reads from 11 walnut leaves according to cDNA libraries. The reads provided a complete de novo transcriptome assembly. Fifteen different transcriptome assemblies were constructed from five different well-known assemblers used in scientific literature with different k-mer lengths (Bridger, BinPacker, SOAPdenovo-Trans, Trinity and SPAdes) as well as two merging approaches (EvidentialGene and Transfuse). Based on the four quality metrics of assembly, the results indicated an efficiency in the process of merging the assemblies after being generated by de novo assemblers. Finally, EvidentialGene was recognized as the best assembler for the de novo assembly of the leaf transcriptome in walnut. Among a total number of 183,191 transcripts which were generated by EvidentialGene, there were 109,413 transcripts capable of protein potential (59.72%) and 104,926 were recognized as ORFs (57.27%). In addition, 79,185 transcripts were predicted to exist with at least one hit to the Pfam database. A number of 3,931 transcription factors were identified by BLAST searching against PlnTFDB. Furthermore, 6,591 of the predicted peptide sequences contained signaling peptides, while 92,704 contained transmembrane domains. Comparison of the assembled transcripts with transcripts of the walnut and published genome assembly for the 'Chandler' cultivar using the BLAST algorithm led to identify a total number of 27,304 and 19,178 homologue transcripts, respectively. De novo transcriptomes in walnut leaves can be developed for the future studies in functional genomics and genetic studies of walnuts