Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology

BackgroundRNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. ResultsThe RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. ConclusionsThis study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments

Quality control assessment of the RNA-Seq data generated from liver and pituitary transcriptome of Hereford bulls using StrandNGS software

Background: Quality control (QC) assessment is the most critical step in the high-throughput RNA-seq data analysis to characterize the in-depth understanding of genome and transcriptome assembling to a given reference genome. It provides not only a quick insight into the RNA-seq data quality to allow early identification of good or bad RNA-seq data samples, but also to verify the alignment QC checks for further essential high-throughput bioinformatics analysis such as, identification of novel genetic variants, differentially expressed genes (DEGs), gene network and metabolic pathways.Method: After isolation of total RNA from liver (n=15) and pituitary gland (n=15) tissues of young Hereford bulls, the pooled total RNA (n=30) were fragmented using GeneRead rRNA depletion kit (Qiagen, Hilden, Germany) and cDNA library preparation were preformed using ScriptSeqTM v2 RNA-Seq library preparation kit (Epicentre, illumina, USA), followed by high-throughput sequencing of combined liver and pituitary transcriptome using MiSeq reagent kit v2 (illumina, USA) to obtain high quality of paired-end RNA-seq reads of 251 base-pairs (bps). In this paper, the QC assessment of obtained RNA-seq raw data as well as post-alignment QC of processed RNA-seq data of combined liver and pituitary transcriptome (n=30) of Hereford bulls were performed using the strand NGS software v1.3 (Agilent; http://www.strand-ngs.com/) data analysis package. The reads were aligned with Bowtie using default settings against both Bull and Cow genome assembly.Results: Using two runs of MiSeq platform, a total of over 60 million paired-end RNA-seq reads were successfully obtained and submitted to NCBI SRA resources (https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=312148). Library complexity plot results revealed 72.02% of duplicate reads with a low library complexity value of 0.28. The pre-alignment QC analysis of raw RNA-seq data revealed the sequence read lengths ranged from 35-251 bp size with more than 50% of all reads with length over 200bp and 10% of reads below 100bp.Conclusion: By testing the RNA-seq methodology on Illumina platform, two MiSeq sequencing runs yielded significantly high quality of 30 million sequencing reads per single MiSeq run. Our initial pre-alignment and post-alignment analysis of RNA-seq data analysis revealed that mapping of the Hereford liver and pituitary gland transcriptome to reference Bos taurus genome was successfully performed, however, more than 50% of all reads with length over 200bp were recovered. Therefore, obtained results concludes that liver and pituitary transcriptome sequencing with rRNA depletion method is less effective than mRNA RNA-seq method

RNA-seq based SNP discovery in liver transcriptome of Polish Landrace pigs

Background: RNA-seq technology is most commonly used in quantitative measurement of gene expression levels and identification of non-annotated transcripts. It is also used for the coding SNPs (cSNPs) discoveries in an efficient and cost-effective way. The aim of this study was to identify the putative genetic cSNPs variants in liver transcriptome of Polish Landrace pigs fed with high and low (normal) omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) diets.Methods: RNA-seq based NGS experiment was performed on Polish Landrace pigs fed with high and low PUFAs diets. Total RNA were isolated from liver tissues of low PUFAs (n=6) and high PUFAs dietary group (n=6) of Polish Landrace pigs. The RNA-seq libraries preparations were performed by mRNA enrichment, mRNA fragmentation, second strand cDNA synthesis, adaptor ligation, size selection and PCR amplification using the illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego CA, USA), followed by NGS sequencing on MiSeq illumina platform. The quality control (QC) of raw RNA-seq data of liver transcriptome was performed using the Trimmomatic and FastQC tools. The paired-end mapping of the liver transcriptome RNA-seq data (n=12) was performed on the reference genome Sus scrofa v.10.2, followed by cSNPs discovery using GATK and SAMtools bioinformatics SNPs caller tools.Results: Two pooled paired-end libraries of 151bp liver transcriptome of Polish Landrace pigs were generated from MiSeq instrument and subsequent Fastq RNA-seq data were submitted to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra). Our study identified 25.3 million paired-end reads: representing 13,509,248 paired-end reads of high PUFAs dietary group and 11,815,696 paired-end reads of low PUFAs dietary group of Polish Landrace pigs liver transcriptome. The SNP discovery results revealed identification of 25909 homozygous and 23290 heterozygous cSNPs in the liver transcriptome of both dietary groups of Polish Landrace pigs. With regards to same or alternative SNPs alleles encoding amino acids regions, a total of 27141 synonymous cSNP and 5989 non-synonymous cSNPs were identified in liver transcriptome representing high PUFAs dietary group. However, a total of 15128 synonymous cSNPs and 3900 non-synonymous cSNPs were identified in liver transcriptome representing low PUFAs dietary groups of Polish Landrace pigs. The identification of single nucleotide variations (SNVs) representing substitutions of all four possibilities (A,T,G,C) were ranged 2872 to 6868 SNVs (high PUFAs) and 2574 to 3654 SNVs (low PUFAs) in the homozygous cSNPs and 2452 to 2678 SNVs (high PUFAs) and 2094 to 2230 SNVs (low PUFAs) in the heterozygous cSNPs of liver transcriptomes of Polish Landrace pigs, respectively.Conclusions: Study concluded that identification of cSNPs dataset representing the liver transcriptome of Polish Landrace pigs fed with a control diet (low) and pigs fed with a PUFAs diet (high) may be helpful to develop a new set of genetic markers for trait-associated studies (viz., growth and metabolic traits) specific to Polish Landrace pig breed. Such cSNP markers eventually can be utilized in the genetic improvement of the pig production traits using the genome-wide association studies (GWAS) and to finally implement on marker assisted selection (MAS) and genomics selection (GS) program in active breeding population of Polish Landrace pigs in Poland

RNA-seq based SNP discovery in gluteus medius muscle of Polish Landrace pigs

BackgroundSingle nucleotide polymorphisms (SNPs) are the well-known molecular markers in genetics and breeding studies applied to veterinary sciences and livestock production. Advancement of next generation sequencing (NGS) provides a high-throughput means of potential putative SNP discovery. The aim of the study was to identify the putative genetic variants in gluteus medius muscle transcriptome of Polish Landrace pigs.MethodsRNA-seq based NGS experiment was performed on Polish Landrace pigs fed with omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) and normal diets. Isolation of total RNA from gluteus medius muscle was performed on low PUFAs (n=6) and High PUFAs dietary group of Polish Landrace pigs. The RNA-seq libraries were constructed by mRNA enrichment, mRNA fragmentation, second strand cDNA synthesis, adaptor ligation, size selection and PCR amplification using the illumina TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego CA, USA), followed by NGS sequencing on MiSeq illumina platform. The quality control of raw RNA-seq data was performed using the Trimmomatic and FastQC tools. High QC paired-end RNA-seq data of gluteus medius muscle transcriptome were mapped to the reference genome Sus scrofa v.10.2. Finally, the SNPs discovery was performed using GATK and SAMtools bioinformatics SNPs caller tools.ResultsThe Fastq RNA-seq data generated from two pooled paired-end libraries (151bp) of gluteus medius muscle tissue of Polish Landrace pigs were submitted to NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra). Study identified a total of 50.5 million paired-end reads (32.5 million low PUFAs dietary group and 18 million reads high PUFAs dietary group) of gluteus medius muscle transcriptome of Polish Landrace pigs. SNP discovery identified a total of 35436 homozygous and 28644 heterozygous cSNPs in gluteus medius muscle transcriptomes representing both dietary groups of Polish Landrace pig. Moreover, a total of 25187 and 5488 cSNP were identified as synonymous SNPs, and 18005 and 4780 cSNP were identified as nonsynonymous SNPs. Finally, single nucleotide variation (SNV) representing substitutions of all four possibilities (A,T,G,C) were identified ranging 2935 to 3227 SNVs (high PUFAs) and 3528 to 3882 SNVs (low PUFAs) for the heterozygous cSNPs and 2712 to 4058 (high PUFAs) and 4169 to 5692 SNVs (low PUFAs) for the heterozygous SNPs in gluteus medius muscle transcriptomes of Polish Landrace pigs.ConclusionsStudy concluded that identification of cSNPs dataset representing the gluteus medius muscle transcriptome of Polish Landrace pigs fed with a control diet (low) and pigs fed with a PUFAs diet (high) may be helpful to develop a new set of genetic markers specific to Polish Landrace pig breed. Such cSNP markers eventually can be utilized in genome-wide association studies (GWAS) and to finally implement on marker assisted selection (MAS) and genomics selection (GS) program in active breeding population of Polish Landrace pigs in Poland

Mean daily plasma concentrations of β-endorphin, leu-enkephalin, ACTH, cortisol, and DHEAS in epileptic patients with complex partial seizures evolving to generalized tonic-clonic seizures

Introduction: A multitude of mechanisms have been implicated in the pathophysiology of epilepsy. Objective: To assess mean daily plasma concentrations of ACTH, cortisol, DHEAS, leu-enkephalin, and beta-endorphin in epileptic patients with complex partial seizures evolving to tonic-clonic in relation to frequency of seizure occurrence (groups with seizure occurrences - several per week and several per year) and duration of the disease (groups less than and more than 10 years). We decided to analyse mean daily values of beta-endorphin and leu-enkephalin because of significant differences in concentrations of these substances in blood during the day. Material and methods: The study was performed on 17 patients (14 males + 3 females; mean age 31.8 yrs) treated with carbamazepine (300-1800 mg/day). The control group consisted of six age-matched healthy volunteers. Blood was collected at 8 a.m., 2 p.m., 8 p.m., and 2 a.m. Intergroup analysis was performed with the use of ANOVA Kruskal-Wallis test. Results: Mean daily concentrations of ACTH and cortisol in the blood of the patients with epilepsy were higher in comparison with those of the healthy volunteers, independently of the frequency of seizures and duration of the disease. Mean daily concentrations of beta-endorphin in the blood of the patients with epilepsy were higher in the groups of patients with more severe clinical course of disease (with more frequently occurring epilepsy seizures and longer duration of the disease) in comparison with healthy subjects. Mean daily concentrations of leu-enkephalin in the blood of the patients with epilepsy were higher in the group of patients with short duration of the disease in comparison with the group with long duration of the disease. Conclusions: 1. Pituitary-adrenal axis hyperactivity is observed in patients with clinically active epilepsy, independently of the frequency of seizures and duration of the disease. 2. Changes in endogenous opioid system activity are related to the clinical activity of epilepsy - beta-endorphin concentrations are connected with frequency of seizures and duration of the disease and leu-enkephalin concentrations with duration of the disease. 3. Endogenous opioid peptides might take part in the neurochemical mechanism of human epilepsy. (Pol J Endocrinol 2010; 61 (1): 103-110)Wstęp: W patofizjologii padaczki uczestniczy nieokreślona ilość mechanizmów. Celem pracy była ocena średniodobowych osoczowych stężeń ACTH, kortyzolu, DHEAS, leu-enkefaliny and β-endorfiny u chorych na padaczkę z napadami częściowymi, złożonymi i wtórnie uogólnionymi, toniczno-klonicznymi w zależności od częstości napadów (grupy z częstością napadów - kilka na tydzień i kilka na rok) i od czasu trwania choroby (grupy 10 lat). Autorzy zdecydowali się na analizę średniodobowych wartości β-endorfiny i leu-enkefaliny z powodu wyraźnych różnic w ich stężeniu we krwi w ciągu doby. Materiał i metody: Badanie przeprowadzono u 17 chorych (14 mężczyzn i 3 kobiety; średni wiek 31,8 lat) leczonych karbamazepiną (300-1800 mg/dzień). Grupa kontrolna składała się z 6 zdrowych ochotników w porównywalnym wieku. Krew pobierano o godzinie 8, 14, 20, 2. W analizie międzygrupowej wykorzystano test ANOVA Kruskala-Wallisa. Wyniki: Średniodobowe stężenia ACTH i kortyzolu we krwi chorych na padaczkę były wyższe w porównaniu ze zdrowymi niezależnie od częstości napadów i czasu trwania choroby. Średniodobowe stężenia β-endorfiny we krwi chorych na padaczkę były wyższe w grupach pacjentów z ciężkim przebiegiem klinicznym choroby (z wysoką częstością napadów i długim czasem trwania choroby) w porównaniu ze zdrowymi. Średniodobowe stężenia leu-enkefaliny we krwi chorych na padaczkę były wyższe w grupie pacjentów z krótkim czasem trwania choroby w porównaniu z grupą z długim czasem trwania choroby. Wnioski: 1. U chorych na padaczkę, niezależnie od częstości napadów i czasu trwania choroby, dochodzi do wzmożonej aktywności osi przysadkowo- nadnerczowej. 2. Zmiany w aktywności endogennego układu opioidowego są związane z kliniczną aktywnością padaczki - stężenia β-endorfiny pozostają w związku z częstością napadów i czasem trwania choroby, a stężenia leu-enkefaliny z czasem trwania choroby. 3. Endogenne peptydy opioidowe mogą uczestniczyć w neurochemicznym mechanizmie padaczki u ludzi. (Endokrynol Pol 2010; 61 (1): 103-110

Comparative Analysis of CpG Sites and Islands Distributed in Mitochondrial DNA of Model Organisms

The information about mtDNA methylation is still limited, thus epigenetic modification remains unclear. The lack of comprehensive information on the comparative epigenomics of mtDNA prompts comprehensive investigations of the epigenomic modification of mtDNA in different species. This is the first study in which the theoretical CpG localization in the mtDNA reference sequences from various species (12) was compared. The aim of the study was to determine the localization of CpG sites and islands in mtDNA of model organisms and to compare their distribution. The results are suitable for further investigations of mtDNA methylation. The analysis involved both strands of mtDNA sequences of animal model organisms representing different taxonomic groups of invertebrates and vertebrates. For each sequence, such parameters as the number, length, and localization of CpG islands were determined with the use of EMBOSS (European Molecular Biology Open Software Suite) software. The number of CpG sites for each sequence was indicated using the newcpgseek algorithm. The results showed that methylation of mtDNA in the analysed species involved mitochondrial gene expression. Our analyses showed that the CpG sites were commonly present in genomic regions including the D-loop, CYTB, ND6, ND5, ND4, ND3, ND2, ND1, COX3, COX2, COX1, ATP6, 16s rRNA, and 12s rRNA. The CpG distribution in animals from different species was diversified. Generally, the number of observed CpG sites of the mitochondrial genome was higher in the vertebrates than in the invertebrates. However, there was no relationship between the frequency of the CpG sites in the mitochondrial genome and the complexity of the analysed organisms. Interestingly, the distribution of the CpG sites for tRNA coding genes was usually cumulated in a larger CpG region in vertebrates. This paper may be a starting point for further research, since the collected information indicates possible methylation regions localized in mtDNA among different species including invertebrates and vertebrates

Oncogenic Mutation BRAF V600E Changes Phenotypic Behavior of THLE-2 Liver Cells through Alteration of Gene Expression

The accumulation of mutations in cancer driver genes, such as tumor suppressors or proto-oncogenes, affects cellular homeostasis. Disturbances in the mechanism controlling proliferation cause significant augmentation of cell growth and division due to the loss of sensitivity to the regulatory signals. Nowadays, an increasing number of cases of liver cancer are observed worldwide. Data provided by the International Cancer Genome Consortium (ICGC) have indicated many alterations within gene sequences, whose roles in tumor development are not well understood. A comprehensive analysis of liver cancer (virus-associated hepatocellular carcinoma) samples has identified new and rare mutations in B-Raf proto-oncogene (BRAF) in Japanese HCC patients, as well as BRAF V600E mutations in French HCC patients. However, their function in liver cancer has never been investigated. Here, using functional analysis and next generation sequencing, we demonstrate the tumorigenic effect of BRAF V600E on hepatocytes (THLE-2 cell line). Moreover, we identified genes such as BMP6, CXCL11, IL1B, TBX21, RSAD2, MMP10, and SERPIND1, which are possibly regulated by the BRAF V600E-mediated, mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. Through several functional assays, we demonstrate that BRAF L537M, D594A, and E648G mutations alone are not pathogenic in liver cancer. The investigation of genome mutations and the determination of their impact on cellular processes and functions is crucial to unraveling the molecular mechanisms of liver cancer development