Methylated ccfDNA from plasma biomarkers of Alzheimer's disease using targeted bisulfite sequencing.

Aim: Noninvasive biomarkers such as methylated ccfDNA from plasma could help to support the diagnosis of Alzheimer's disease (AD). Methods: A targeted sequencing protocol was developed to identify candidate biomarkers of AD in methylated ccfDNA extracted from plasma. Results: The authors identified differentially methylated CpGs, regions of which were the same as those identified in previous AD studies. Specifically, a differentially methylated CpG of the LHX2 gene previously identified in a plasma study of AD was replicated in the study. The MBP and DUSP22 regions have been identified in other brain studies of AD and in the authors' study. Conclusion: Although these biomarkers must be validated in other cohorts, methylated ccfDNA could be a relevant noninvasive biomarker in AD.

Currently, the diagnosis of Alzheimer's disease (AD) is based on symptoms and medical imaging, and definitive clinical diagnosis is only possible postmortem. The identification of noninvasive biomarkers such as methylated ccfDNA is crucial for the diagnosis, prognosis and monitoring of AD. However, the analysis of ccfDNA from plasma is a challenge because it is highly fragmented and present in low amounts and originates from various tissues. The authors developed a targeted sequencing protocol using genes previously reported in AD literature (brain, blood and plasma) to identify potential noninvasive biomarkers in plasma. The authors identified positions identical to those in the literature as well as potential novel sites located in the promoter, exon and intron regions of these genes. Although these results must be validated in a large cohort, methylated ccfDNA could be a useful noninvasive biomarker for AD.

PIntMF: Penalized Integrative Matrix Factorization method for multi-omics data.

MOTIVATION: It is more and more common to perform multi-omics analyses to explore the genome at diverse levels and not only at a single level. Through integrative statistical methods, multi-omics data have the power to reveal new biological processes, potential biomarkers and subgroups in a cohort. Matrix factorization (MF) is an unsupervised statistical method that allows a clustering of individuals, but also reveals relevant omics variables from the various blocks. RESULTS: Here, we present PIntMF (Penalized Integrative Matrix Factorization), an MF model with sparsity, positivity and equality constraints. To induce sparsity in the model, we used a classical Lasso penalization on variable and individual matrices. For the matrix of samples, sparsity helps in the clustering, while normalization (matching an equality constraint) of inferred coefficients is added to improve interpretation. Moreover, we added an automatic tuning of the sparsity parameters using the famous glmnet package. We also proposed three criteria to help the user to choose the number of latent variables. PIntMF was compared with other state-of-the-art integrative methods including feature selection techniques in both synthetic and real data. PIntMF succeeds in finding relevant clusters as well as variables in two types of simulated data (correlated and uncorrelated). Next, PIntMF was applied to two real datasets (Diet and cancer), and it revealed interpretable clusters linked to available clinical data. Our method outperforms the existing ones on two criteria (clustering and variable selection). We show that PIntMF is an easy, fast and powerful tool to extract patterns and cluster samples from multi-omics data. AVAILABILITY AND IMPLEMENTATION: An R package is available at https://github.com/mpierrejean/pintmf. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA.

Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnosis, prognosis and monitoring treatment of disease. However, a sensitive and specific whole-genome sequencing (WGS) method is required to identify novel genetic variations (i.e., SNVs, CNVs and INDELS) on ccfDNA that can be used as clinical biomarkers. In this article, five WGS methods were compared: ThruPLEX Plasma-seq, QIAseq cfDNA All-in-One, NEXTFLEX Cell Free DNA-seq, Accel-NGS 2 S PCR FREE DNA and Accel-NGS 2 S PLUS DNA. The Accel PCR-free kit did not produce enough material for sequencing. The other kits had significant common number of SNVs, INDELs and CNVs and showed similar results for SNVs and CNVs. The detection of variants and genomic signatures depends more upon the type of plasma sample rather than the WGS method used. Accel detected several variants not observed by the other kits. ThruPLEX seemed to identify more low-abundant SNVs and SNV signatures were similar to signatures observed with the QIAseq kit. Accel and NEXTFLEX had similar CNV and SNV signatures. These results demonstrate the importance of establishing a standardized workflow for identifying non-invasive candidate biomarkers. Moreover, the combination of variants discovered in ccfDNA using WGS has the potential to identify enrichment pathways, while the analysis of signatures could identify new subgroups of patients.

Clustering and variable selection evaluation of 13 unsupervised methods for multi-omics data integration.

Recent advances in NGS sequencing, microarrays and mass spectrometry for omics data production have enabled the generation and collection of different modalities of high-dimensional molecular data. The integration of multiple omics datasets is a statistical challenge, due to the limited number of individuals, the high number of variables and the heterogeneity of the datasets to integrate. Recently, a lot of tools have been developed to solve the problem of integrating omics data including canonical correlation analysis, matrix factorization and SM. These commonly used techniques aim to analyze simultaneously two or more types of omics. In this article, we compare a panel of 13 unsupervised methods based on these different approaches to integrate various types of multi-omics datasets: iClusterPlus, regularized generalized canonical correlation analysis, sparse generalized canonical correlation analysis, multiple co-inertia analysis (MCIA), integrative-NMF (intNMF), SNF, MoCluster, mixKernel, CIMLR, LRAcluster, ConsensusClustering, PINSPlus and multi-omics factor analysis (MOFA). We evaluate the ability of the methods to recover the subgroups and the variables that drive the clustering on eight benchmarks of simulation. MOFA does not provide any results on these benchmarks. For clustering, SNF, MoCluster, CIMLR, LRAcluster, ConsensusClustering and intNMF provide the best results. For variable selection, MoCluster outperforms the others. However, the performance of the methods seems to depend on the heterogeneity of the datasets (especially for MCIA, intNMF and iClusterPlus). Finally, we apply the methods on three real studies with heterogeneous data and various phenotypes. We conclude that MoCluster is the best method to analyze these omics data. Availability: An R package named CrIMMix is available on GitHub at https://github.com/CNRGH/crimmix to reproduce all the results of this article.

Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies.

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes. Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR). E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

AIM: The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. MATERIALS & METHODS: Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. RESULTS: E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. CONCLUSION: E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Whole-Genome Bisulfite Sequencing for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and disease-associated investigations. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered as the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here we provide two detailed protocols based on commercial kits for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. If only DNA methylation is of interest, sequencing libraries can be constructed from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For samples with significant levels of hydroxymethylation such as stem cells or brain tissue, we describe the protocol of oxidative bisulfite sequencing (OxBs-seq), which in its current version uses a post-bisulfite adaptor tagging (PBAT) approach. Two methylomes need to be generated: a classic methylome following bisulfite conversion and analyzing both methylated and hydroxymethylated cytosines and a methylome analyzing only methylated cytosines, respectively. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocols have been successfully applied to different human samples and yield robust and reproducible results.

Whole-Genome Bisulfite Sequencing Using the Ovation® Ultralow Methyl-Seq Protocol.

The analysis of genome-wide epigenomic alterations including DNA methylation has become a subject of intensive research for many complex diseases. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies can be considered the gold standard for a comprehensive and quantitative analysis of cytosine methylation throughout the genome. Several approaches including tagmentation- and post bisulfite adaptor tagging (PBAT)-based WGBS have been devised. Here, we provide a detailed protocol based on a commercial kit for the preparation of libraries for WGBS from limited amounts of input DNA (50-100 ng) using the classical approach of WGBS by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. The converted library is then amplified with an optimal number of PCR cycles to ensure high sequence diversity and low duplicate rates. Spike-in of unmethylated DNA allows for the precise estimation of bisulfite conversion rates. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human samples as well as DNA extracted from plant tissues and yields robust and reproducible results.

COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy.

Recurrent NRAS mutations in pulmonary Langerhans cell histiocytosis.

The mitogen-activated protein kinase (MAPK) pathway is constantly activated in Langerhans cell histiocytosis (LCH). Mutations of the downstream kinases BRAF and MAP2K1 mediate this activation in a subset of LCH lesions. In this study, we attempted to identify other mutations which may explain the MAPK activation in nonmutated BRAF and MAP2K1 LCH lesions.We analysed 26 pulmonary and 37 nonpulmonary LCH lesions for the presence of BRAF, MAP2K1, NRAS and KRAS mutations. Grossly normal lung tissue from 10 smoker patients was used as control. Patient spontaneous outcomes were concurrently assessed.BRAF(V600E) mutations were observed in 50% and 38% of the pulmonary and nonpulmonary LCH lesions, respectively. 40% of pulmonary LCH lesions harboured NRAS(Q61K) (/R) mutations, whereas no NRAS mutations were identified in nonpulmonary LCH biopsies or in lung tissue control. In seven out of 11 NRAS(Q61K) (/R)-mutated pulmonary LCH lesions, BRAF(V600) (E) mutations were also present. Separately genotyping each CD1a-positive area from the same pulmonary LCH lesion demonstrated that these concurrent BRAF and NRAS mutations were carried by different cell clones. NRAS(Q61K) (/R) mutations activated both the MAPK and AKT (protein kinase B) pathways. In the univariate analysis, the presence of concurrent BRAF(V600E) and NRAS(Q61K) (/R) mutations was significantly associated with patient outcome.These findings highlight the importance of NRAS genotyping of pulmonary LCH lesions because the use of BRAF inhibitors in this context may lead to paradoxical disease progression. These patients might benefit from MAPK kinase inhibitor-based treatments.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma.

Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnostics, and prediction and monitoring of treatment response, but its amount is usually limited. Therefore, the choice of methods to extract and characterize ccfDNA is crucial. In the current study, we performed the most comprehensive comparison of methods for ccfDNA extraction (11 methods), quantification (3 methods), and estimation of the integrity index (2 methods) from small quantities of different kinds of plasma. The QIAamp® Circulating Nucleic Acid Kit and the Norgen Plasma/Serum Circulating DNA Purification Mini Kit showed the best accuracy and reproducibility, but the Norgen kit allowed to extract a higher amount of ccfDNA. This workflow provides a reliable protocol for the multiple applications of ccfDNA in biomedicine.

A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras.

RATIONALE: High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group. METHODS: Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell. RESULTS: Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4(-) product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)-BiH](-), [ai-BiH](-), wj(-), [wj, (ai-BiH)](-) (if Bi ≠ T) as internal product ion, and more rarely [wj-BiH](-). The absence of the majority of the [ai-BiH](-) series although the wj (-) series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj(-) series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced. CONCLUSIONS: This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances.

High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

Here, we describe a high-throughput, single-tube, allele-specific ribonucleotide analog pyrophosphorolysis-activated polymerization (ribo-PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele-selective 5'-tagged primers, a reverse primer, one nucleotide in the ribo-form (90-100%), the other nucleotides in the deoxy-form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele-selective primers have a 5' repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo-PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo-PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo-PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo-PAP PCR.

Ribo-polymerase chain reaction--a facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing.

We describe ribo-polymerase chain reaction (PCR), a method for the preparation of chimeric RNA/DNA. The RNA/DNA chimeric nucleic acids are generated directly from genomic DNA starting templates with two locus-specific primers, three nucleotides in their deoxy form and the fourth in its ribo form, a DNA polymerase capable of incorporating ribo bases, a suitable buffer, and thermal cycling. We have applied ribo-PCR to resequence DNA by directly fragmenting the RNA/DNA chimeras with alkali and analyzing the fragments by mass spectrometry (MS). Mass fingerprint is used to identify deviations from the reference sequence. This method readily detects homozygous sequence deviations as well as heterozygous positions directly from genomic DNA samples. With the high-throughput capability of MS, this facile method is well suited for screening DNA sequences of limited regions of the genome in a large number of individuals. It can also be used to sequence multiple distant genomic loci in a single reaction. This novel ribo-PCR resequencing protocol was applied to different genomic loci involving nitric oxide synthase 1 (NOS1) and H19 in 30 individuals and SLCO1B1 in 95 individuals.

DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras.

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3' of all ribonucleotides to form a terminal 3' phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry.

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3' terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.