Can long-read sequencing tackle the barriers, which the next-generation could not? A review.

The large-scale heterogeneity of genetic diseases necessitated the deeper examination of nucleotide sequence alterations enhancing the discovery of new targeted drug attack points. The appearance of new sequencing techniques was essential to get more interpretable genomic data. In contrast to the previous short-reads, longer lengths can provide a better insight into the potential health threatening genetic abnormalities. Long-reads offer more accurate variant identification and genome assembly methods, indicating advances in nucleotide deflect-related studies. In this review, we introduce the historical background of sequencing technologies and show their benefits and limits, as well. Furthermore, we highlight the differences between short- and long-read approaches, including their unique advances and difficulties in methodologies and evaluation. Additionally, we provide a detailed description of the corresponding bioinformatics and the current applications.

Patterns of Somatic Variants in Colorectal Adenoma and Carcinoma Tissue and Matched Plasma Samples from the Hungarian Oncogenome Program.

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.

Methodological and Biological Factors Influencing Global DNA Methylation Results Measured by LINE-1 Pyrosequencing Assay in Colorectal Tissue and Liquid Biopsy Samples.

Long interspersed nuclear element 1 (LINE-1) bisulfite pyrosequencing is a widely used technique for genome-wide methylation analyses. We aimed to investigate the effects of experimental and biological factors on its results to improve the comparability. LINE-1 bisulfite pyrosequencing was performed on colorectal tissue (n = 222), buffy coat (n = 39), and plasma samples (n = 9) of healthy individuals and patients with colorectal tumors. Significantly altered methylation was observed between investigated LINE-1 CpG positions of non-tumorous tissues (p ≤ 0.01). Formalin-fixed, paraffin-embedded biopsies (73.0 ± 5.3%) resulted in lower methylation than fresh frozen samples (76.1 ± 2.8%) (p ≤ 0.01). DNA specimens after long-term storage showed higher methylation levels (+3.2%, p ≤ 0.01). In blood collection tubes with preservatives, cfDNA and buffy coat methylation significantly changed compared to K3EDTA tubes (p ≤ 0.05). Lower methylation was detected in older (>40 years, 76.8 ± 1.7%) vs. younger (78.1 ± 1.0%) female patients (p ≤ 0.05), and also in adenomatous tissues with MTHFR 677CT, or 1298AC mutations vs. wild-type (p ≤ 0.05) comparisons. Based on our findings, it is highly recommended to consider the application of standard DNA samples in the case of a possible clinical screening approach, as well as in experimental research studies.

A Detailed Overview About the Single-Cell Analyses of Solid Tumors Focusing on Colorectal Cancer.

In recent years, the evolution of the molecular biological technical background led to the widespread application of single-cell sequencing, a versatile tool particularly useful in the investigation of tumor heterogeneity. Even 10 years ago the comprehensive characterization of colorectal cancers by The Cancer Genome Atlas was based on measurements of bulk samples. Nowadays, with single-cell approaches, tumor heterogeneity, the tumor microenvironment, and the interplay between tumor cells and their surroundings can be described in unprecedented detail. In this review article we aimed to emphasize the importance of single-cell analyses by presenting tumor heterogeneity and the limitations of conventional investigational approaches, followed by an overview of the whole single-cell analytic workflow from sample isolation to amplification, sequencing and bioinformatic analysis and a review of recent literature regarding the single-cell analysis of colorectal cancers.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content.

BACKGROUND: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. METHODS: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40). RESULTS: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). CONCLUSION: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced γ-H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Diagnostic and prognostic potential of tissue and circulating long non-coding RNAs in colorectal tumors.

Long non-coding RNAs (lncRNAs) are members of the non-protein coding RNA family longer than 200 nucleotides. They participate in the regulation of gene and protein expression influencing apoptosis, cell proliferation and immune responses, thereby playing a critical role in the development and progression of various cancers, including colorectal cancer (CRC). As CRC is one of the most frequently diagnosed malignancies worldwide with high mortality, its screening and early detection are crucial, so the identification of disease-specific biomarkers is necessary. LncRNAs are promising candidates as they are involved in carcinogenesis, and certain lncRNAs (e.g., CCAT1, CRNDE, CRCAL1-4) show altered expression in adenomas, making them potential early diagnostic markers. In addition to being useful as tissue-specific markers, analysis of circulating lncRNAs (e.g., CCAT1, CCAT2, BLACAT1, CRNDE, NEAT1, UCA1) in peripheral blood offers the possibility to establish minimally invasive, liquid biopsy-based diagnostic tests. This review article aims to describe the origin, structure, and functions of lncRNAs and to discuss their contribution to CRC development. Moreover, our purpose is to summarise lncRNAs showing altered expression levels during tumor formation in both colon tissue and plasma/serum samples and to demonstrate their clinical implications as diagnostic or prognostic biomarkers for CRC.

Comparison of Automated and Manual DNA Isolation Methods for DNA Methylation Analysis of Biopsy, Fresh Frozen, and Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples.

Automated DNA isolation can decrease hands-on time in routine pathology. Our aim was to apply automated DNA isolation and perform DNA methylation analyses. DNA isolation was performed manually from fresh frozen (CRC = 10, normal = 10) specimens and colonic biopsies (CRC = 10, healthy = 10) with QIAamp DNA Mini Kit and from FFPE blocks (CRC = 10, normal = 10) with QIAamp DNA FFPET Kit. Automated DNA isolation was performed with MagNA Pure DNA and Viral NA SV kit on MagNA Pure 96 system. DNA methylation of MAL, SFRP1, and SFRP2 were analyzed with methylation-specific high-resolution melting analysis. Yield of automatically isolated samples was equal in fresh frozens and significantly lower compared to manually isolated biopsy and FFPE samples. OD260/280 of fresh frozen and biopsy samples were similar after both isolations, automated isolation resulted in lower purity in FFPE samples. Both protocols resulted in similar OD260/230 from fresh frozens, automated isolation method was superior in biopsies and manual protocol in FFPE samples. DNA methylation of biopsies, fresh frozen samples were highly similar after both methods, results of automatically and manually isolated FFPE samples were different. Automated DNA isolation from fresh frozen samples can be suitable for high-throughput laboratories.

Complete genes may pass from food to human blood.

Our bloodstream is considered to be an environment well separated from the outside world and the digestive tract. According to the standard paradigm large macromolecules consumed with food cannot pass directly to the circulatory system. During digestion proteins and DNA are thought to be degraded into small constituents, amino acids and nucleic acids, respectively, and then absorbed by a complex active process and distributed to various parts of the body through the circulation system. Here, based on the analysis of over 1000 human samples from four independent studies, we report evidence that meal-derived DNA fragments which are large enough to carry complete genes can avoid degradation and through an unknown mechanism enter the human circulation system. In one of the blood samples the relative concentration of plant DNA is higher than the human DNA. The plant DNA concentration shows a surprisingly precise log-normal distribution in the plasma samples while non-plasma (cord blood) control sample was found to be free of plant DNA.