GC Distribution Research Articles

QTL Mapping and Genetic Map for the Ornamental Sunflower in China

Quantitative trait locus (QTL) mapping and genetic map are of great significance for ornamental sunflowers in China. In this study, a total of 956.50 Mbp data were obtained, the average Q30 was 93.76%, the average GC content was 42.43%, and the GC distribution of the parents and F2 population of the ornamental sunflowers was normal. At the same time, the double-end comparison efficiency of control data was 90.28%, and the enzyme digestion efficiency was 92.01%. The Specific-Locus Amplified Fragment (SLAF) library construction was normal. Furthermore, a total of 734,893 SLAF markers were obtained, among which 127,855 were polymorphic SLAF markers and 38,908 could be used for genetic map construction, and the effective polymorphism of the parents was 5.29%. Moreover, we constructed a total of 17 linkage groups, with 6181 markers in the QTL mapping, the total map distance was 2608.66 cM, the marker integrity in the figure above was 99%, the proportion of double exchange was 0.05, the sequencing depth of the parents was 42.455 × , and the progeny was 9.24 × . The relationship of traits (plant height, stem diameter, disk diameter, number of petals, leaf number, stigma color, petal color, petiole color) and QTL mapping was closely related to show the best of ornamental effect.

Virus impacted community adaptation in oligotrophic groundwater environment revealed by Hi-C coupled metagenomic and viromic study

Viruses play a crucial role in microbial mortality, diversity and biogeochemical cycles. Groundwater is the largest global freshwater and one of the most oligotrophic aquatic systems on Earth, but how microbial and viral communities are shaped in this special habitat is largely unexplored. In this study, we collected groundwater samples from 23 to 60 m aquifers at Yinchuan Plain, China. In total, 1920 non-reductant viral contigs were retrieved from metagenomes and viromes constructed by Illumina and Nanopore hybrid sequencing. Only 3% of them could be clustered with known viruses, most of which were Caudoviricetes. Coupling 1.2 Tb Hi-C sequencing with CRISPR matching and homology search, we connected 469 viruses with their hosts while some viral clusters presented a broad-host-range trait. Meanwhile, a large proportion of biosynthesis related auxiliary metabolism genes were identified. Those characteristics might benefit viruses for a better survival in this special oligotrophic environment. Additionally, the groundwater virome showed genomic features distinct from those of the open ocean and wastewater treatment facilities in GC distribution and unannotated gene compositions. This paper expands the current knowledge of the global viromic records and serves as a foundation for a more thorough understanding of viruses in groundwater.

Identification of DNA methylation and genetic alteration simultaneously from a single blood biopsy.

High-throughput sequencing of blood cell-free DNA (cfDNA) techniques offer an opportunity to characterize and monitor cancer rapidly in a non-invasive and real-time manner. Nonetheless, there lacks a tool within therapeutic arsenal to identify multi-omics alterations simultaneously from a single biopsy. In current times, bisulfite-based sequencing detects 5mC and 5hmC at single-base resolution is the golden standard of DNA methylation, while the degradation of DNA and biased sequencing data are the problems of this method. To identify the consistency analysis of methylation and genetic variation with single library, we presented a platform detecting multi-omics data simultaneously from a single blood biopsy using bisulfite-free method of genomic methylation sequencing (GM-seq) mediated by TET enzyme. We detected methylomic and genetic changes simultaneously from a single blood biopsy in NA12878 and randomly chose ten blood biopsies from colorectal cancer or lung cancer patients to validate the ability of GM-seq. Similar cytosine methylation level between whole genome bisulfite sequencing (WGBS) and GM-seq were identified in NA12878. Moreover, longer insert size, CpGs coverage and GC distribution were outperformed than WGBS. In addition, the comparison of the single nucleotide polymorphism (SNP), insertion-deletion (Indel) and copy number variation (CNV) in NA12878 or ctDNA from liver cancer between GM-seq and whole genome sequencing (WGS) show a good consistency, indicating that this method is feasible for detecting genetic variation in blood. In conclusion, our work demonstrated a method for identification of the methylated modification and genetic variations simultaneously from a single blood biopsy.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5′ tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Research on the statistical characteristic of freak waves based on observed wave data

Freak waves are characterized by high wave, great destructive power, abrupt behavior, and a significant threat to offshore structures. In this study, the observed wave data of multiple sea areas are utilized, the relation between the occurrence of freak waves at real sea states and the statistical characteristics of wave trains are investigated, and the probable reasons for freak wave generation at real sea conditions is explored. It can be found that for the wave trains with higher kurtosis, the occurrence probability of huge wave height is underestimated by the Rayleigh distribution. It can be accurately predicted by the MER distribution and GC distribution. Generally, the occurrence probability of freak wave increases linearly with the increase of kurtosis from the statistical results, and the MER model is capable of describing the relation between occurrence probability of freak wave and kurtosis. Moreover, the generation types of freak waves are analyzed based on the wavelet energy spectrum and time process of wave surface for typical freak waves in the observation data. Freak waves at real sea states are attributed to the modulation instability and wave energy superposition.

Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA.

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Molecular characterization of small indigenous fish species (SIS) of Bangladesh through DNA barcodes

The mitochondrial DNA gene cytochrome oxidase subunit I (COI) has been adopted as a global bioidentification system for animals. This study represents the comprehensive molecular identification of small indigenous fish species (SIS) of Bangladesh assessed by DNA barcoding. DNA barcodes were generated from 81 SIS species belongs to the orders Clupeiformes, Cypriniforms, Siluriformes, Perciformes, Synbranchiformes, Beloniformes and Tetraodontiformes representing 55 genera and 24 families. For all the samples, %G were significantly lowered compared to other nucleotides and %GC compared to %AT. Also, a significantly lowered GC content was observed in second and third codon position compared to the first codon position in all samples. The average Kimura two-parameter (K2P) distances within genera, families, and orders were 15.83%, 19.14%, and 25.07%, respectively. The minimum and maximum K2P distance based genetic divergences were 0.19% and 57.14% respectively. Members of Cypriniformes, Siluriformes, and Perciformes were clustered separately in the neighbour-joining (NJ) tree. Nucleotide composition, GC distribution across codon positions, K2P distance, genetic divergence, and phylogenetic analyses reveal that these freshwater SIS fishes are genetically very diverse. Along with morphological data, we have confirmed the existence of seven new records of SIS fishes in Bangladesh using such barcode approach. These findings suggest that fishes can be discriminated using these barcode data without any confusion.

Correlation between GDF-15 gene polymorphism and the formation of collateral circulation in acute ST-elevation myocardial infarction.

To explore the correlation between growth differentiation factor 15 (GDF-15) -3148C/G polymorphism and the formation of collateral circulation in acute ST-elevation myocardial infarction (STEMI) in Han population of Taiyuan area. The present study included 92 STEMI patients and 56 normal controls based on coronary angiography; STEMI group was divided into collateral group and non-collateral group according to Rentrop's grading method. Polymerase chain reaction (PCR) and DNA sequencing methods were used to detect and analyze the GDF-15 -3148C/G polymorphism in all participants. There was significant difference in GDF-15 -3148C/G CC and GC distribution between STEMI group and control group (p=0.009); the allele frequencies between these two groups were also significant different (p=0.016); and the risk genotype for STEMI was CC with increased OR=2.660. For STEMI group, GDF-15 -3148C/G CC and GC distribution was also significantly different between patients with and without collateral (p=0.048), and CC genotype significantly promote the formation of collateral circulation. However, there were no significant differences in allele frequencies between these two subgroups of STEMI. There was correlation between GDF-15-3148C/G polymorphism and the formation of collateral circulation in patients with acute STEMI.

Crack propagation along interface having randomly fluctuating mechanical properties during DCB test finite difference implementation – Evaluation of G distribution with effective crack length technique

Abstract A finite difference numerical scheme is implemented to study the crack initiation and propagation along a Double Cantilever Beam specimen assuming either elastic-brittle interface or elastic perfectly plastic behavior. The finite difference calculations are first compared to known analytic solution to evidence the benefits and limits of this numerical method. Then randomly fluctuating interface properties along the crack propagation path is considered to investigate the effect of variability on the force versus opening displacement evolution measured at a macroscopic scale. The local fluctuation of the critical Strain Energy Release Rate (SERR), Gc, could be estimated using the effective crack length technique. However, local minima of the critical strain energy release rate induce unstable crack propagation so that the weakest regions of the interface are not probed. Also, fluctuation of interface properties induce distortion of Gc distribution which could lead to non-conservative evaluation of the interface toughness.

Codon Usage Bias and Determining Forces in Taenia solium Genome.

The tapeworm Taenia solium is an important human zoonotic parasite that causes great economic loss and also endangers public health. At present, an effective vaccine that will prevent infection and chemotherapy without any side effect remains to be developed. In this study, codon usage patterns in the T. solium genome were examined through 8,484 protein-coding genes. Neutrality analysis showed that T. solium had a narrow GC distribution, and a significant correlation was observed between GC12 and GC3. Examination of an NC (ENC vs GC3s)-plot showed a few genes on or close to the expected curve, but the majority of points with low-ENC (the effective number of codons) values were detected below the expected curve, suggesting that mutational bias plays a major role in shaping codon usage. The Parity Rule 2 plot (PR2) analysis showed that GC and AT were not used proportionally. We also identified 26 optimal codons in the T. solium genome, all of which ended with either a G or C residue. These optimal codons in the T. solium genome are likely consistent with tRNAs that are highly expressed in the cell, suggesting that mutational and translational selection forces are probably driving factors of codon usage bias in the T. solium genome.

Probing plant evolution by GC content

Plant Genomics Scientists use GC content (that is, the percentage of guanine or cytosine residues in a genome) as a proxy to measure many elements relating to gene evolution. Within the major group of flowering plants called monocots, which includes many agriculturally important species, the GC content of the genomes of grasses decreases from the 5' to 3' end of the gene. In order to better understand how the distribution of GC content evolved in monocots, Clement et al. examined orthologous genes across 10 monocot species. They found that the specific pattern of GC distribution seen in grasses is in fact not grassspecific—it is ancestral to the monocots. Genome Biol. Evol. 10.1093/gbe/evu278 (2014).

Comparative analysis of codon usage patterns in chloroplast genomes of the Asteraceae family

Codon usage bias (CUB) is an important evolutionary feature in a genome and has been widely documented from prokaryotes to eukaryotes. However, the significance of CUB in the Asteraceae family has not been well understood, with no Asteraceae species having been analyzed for this characteristic. Here, we use bioinformatics approaches to comparatively analyze the general patterns and influencing factors of CUB in five Asteraceae chloroplast (cp) genomes. The results indicated that the five genomes had similar codon usage patterns, showing a strong bias towards a high representation of NNA and NNT codons. Neutrality analysis showed that these cp genomes had a narrow GC distribution and no significant correlation was observed between GC12 and GC3. Parity Rule 2 (PR2) plot analysis revealed that purines were used more frequently than pyrimidines. Effective number of codons (ENc)-plot analysis showed that most genes followed the parabolic line of trajectory, but several genes with low ENc values lying below the expected curve were also observed. Furthermore, correspondence analysis of relative synonymous codon usage (RSCU) yielded a first axis that explained only a partial amount of variation of codon usage. These findings suggested that both natural selection and mutational bias contributed to codon bias, while selection was the major force to shape the codon usage in these Asteraceae cp genomes. Our study, which is the first to investigate codon usage patterns in Asteraceae plastomes, will provide helpful information about codon distribution and variation in these species, and also shed light on the genetic and evolutionary mechanisms of codon biology within this family.

A tale of three next generation sequencing platforms: comparison of Ion torrent, pacific biosciences and illumina MiSeq sequencers

BackgroundNext generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy.ResultsSequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.ConclusionsAll three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

English

  The aim of this study was to present a new situation in which a relatively single short PCR-product might show two separate peaks with sequence specific shapes at the dissociation curve. SYBR-Green I real-time RT-PCR was performed on Lhcgr-gene transcripts in rats. Different programs were used for melting curve simulation and estimating Tm. Statistical tests were performed to determine whether two peaks at the dissociation curve were belonging to a single template. A bimodal melting curve was observed in real-time RT-PCR on a short segment (169 bp) of Lhcgr gene with a single band in gel electrophoresis. Sequencing of the Cloned PCR-product was compatible with template sequence. Real-time PCR using the vector conveying interested sequence, showed again two peaks at dissociation curve. The GC-content of first 100 bases (75%) and last 69 bases (42%) were significantly different. DNA melting simulation programs also confirmed the bimodal pattern, although, their height and wideness were different to actual peaks. Due to the asymmetric GC distribution effect on dissociation curve in short sequences, it is highly recommended to use DNA melting simulation programs to predict the number of peaks in the melting curve when designating primers; however, predicted peak shapes are not always accurate.   Key words: Asymmetric GC distribution, bimodal melting curve, DNA melting simulation, SYBR-green I real-time PCR.

The effect of GC distribution on melting curve analysis in real-time PCR

The effect of GC distribution on melting curve analysis in real-time PCR

Age and metallicity of compact stellar systems in Virgo and Fornax

For high signal-to-noise ratio GMOS-N and AAOmega spectra, we have measured line-strength indices in the Lick/IDS system for several luminous and spatially dispersed compact stellar systems (CSSs) located in the Virgo and Fornax galaxy clusters. We estimate their [alpha/Fe] ratios, ages and metallicities using a simple (single-burst) stellar population model We confirm that the Virgo core region luminous CSSs (M(bj) <= -10.5) contain ancient stellar populations with subsolar total metallicities, suggesting that they comprise the bright tail of M87's GC distribution. The two Virgo intracluster globular clusters have ages and metallicities consistent with the cluster core CSSs. Two Fornax luminous CSSs also have ancient stellar populations but are at the upper end of the Virgo CSS metallicity range, while the third (UCD3) appears to be relatively young, metal-rich and with a core + halo radial profile. Our results suggest that Fornax may contain an extra population of luminous CSSs formed more recently than the ancient GC-like systems found in both clusters.

Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.

Insights into the Musa genome: Syntenic relationships to rice and between Musa species

BackgroundMusa species (Zingiberaceae, Zingiberales) including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome.ResultsWe produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales) orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya.ConclusionThese results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic traits for breeding purposes.

Biased Distributions and Decay of Long Interspersed Nuclear Elements in the Chicken Genome

The genomes of birds are much smaller than mammalian genomes, and transposable elements (TEs) make up only 10% of the chicken genome, compared with the 45% of the human genome. To study the mechanisms that constrain the copy numbers of TEs, and as a consequence the genome size of birds, we analyzed the distributions of LINEs (CR1's) and SINEs (MIRs) on the chicken autosomes and Z chromosome. We show that (1) CR1 repeats are longest on the Z chromosome and their length is negatively correlated with the local GC content; (2) the decay of CR1 elements is highly biased, and the 5'-ends of the insertions are lost much faster than their 3'-ends; (3) the GC distribution of CR1 repeats shows a bimodal pattern with repeats enriched in both AT-rich and GC-rich regions of the genome, but the CR1 families show large differences in their GC distribution; and (4) the few MIRs in the chicken are most abundant in regions with intermediate GC content. Our results indicate that the primary mechanism that removes repeats from the chicken genome is ectopic exchange and that the low abundance of repeats in avian genomes is likely to be the consequence of their high recombination rates.

Genetic code symmetry and efficient design of GC-constrained coding sequences

Cloning of long DNA sequences (40-60 bases) into phage display libraries using polymerase chain reaction (PCR) is a low efficiency process, in which PCR is used to incorporate a DNA insert, coding for a certain peptide, into the amplified sequence. The PCR efficiency in this process is strongly affected by the distribution of G-C bases in the amplified sequence. As any DNA insert coding for the target peptide may be attempted, there is a flexibility in choosing part of the amplified sequence. Since the number of inserts coding for the same peptide is exponential in the peptide length, a computational problem naturally arises--that of efficiently finding an insert, whose parameters are optimal for PCR cloning. The GC distribution requirements are formulated as a search problem. We developed an efficient, linear time 'one pass' algorithm for this problem. Interestingly, our algorithm strongly relies on an interesting symmetry, which we observed in the standard genetic code. Most non-standard genetic codes examined possess this symmetry as well, yet some do not. We generalize the search problem and consider the case of a non-standard, or arbitrary, genetic code where this symmetry does not necessary hold. We solve the generalized problem in polynomial, but nonlinear, time. An implementation of the proposed algorithm is available upon request from the authors.