Fungal DNA extraction for Nanopore sequencing v1

This protocol is intended for extraction of high molecular weight DNA from fungal samples.

An optimized method for high-quality DNA extraction medicinal fungi Mycoleptodonoides aitchisonii for whole genome sequencing

The extraction of pure high molecular weight DNA and collecting genomic DNA from fungi is difficult. This is because filamentous fungi has a sturdy cell wall, high protein and polysaccharide levels resistant to the usual DNA extraction procedures. A low-cost and reliable DNA extraction method was designed from Mycoleptodonoides. aitchisonii fit for whole-genome sequencing for identification and mapping of the A mating-type gene. Mycoleptodonoides. aitchisonii belongs to the Climacodontaceae family has pharmaceutical activities. In the present study, the mycelia of M. aitchisonii, which grew from the different concentrations of malt extract and minimal liquid media, have been compared systematically to determine the DNA extraction procedure resulted in DNA concentration with excellent purity and quality. The best protocol that resulted in good quality DNA was further validated using polymerase chain reaction amplification with a specific primer to amplify the homeodomain protein (Mahd2-18) gene that encodes a transcription factor. The method proposed DNA extraction using CTAB (Cetyl Trimethyl Ammonium Bromide) method and purify by commercial kit from mycelium grown in the minimal liquid media give the best result with the high concentration of DNA for whole-genome sequencing by Next Generation Sequencing and other applications.

SVJAM: Joint Analysis of Structural Variants Using Linked Read Sequencing Data

Linked-read whole genome sequencing methods, such as the 10x Chromium, attach a unique molecular barcode to each high molecular weight DNA molecule. The samples are then sequenced using short-read technology. During analysis, sequence reads sharing the same barcode are aligned to adjacent genomic locations. The pattern of barcode sharing between genomic regions allows the discovery of large structural variants (SVs) in the range of 1 Kb to a few Mb. Most SV calling methods for these data, such as LongRanger, analyze one sample at a time and often produces inconsistent results for the same genomic location across multiple samples. We developed a method, SVJAM, for joint calling of SVs, using data from 152 members of the BXD family of recombinant inbred strains of mice. Our method first collects candidate SV regions from single sample analysis, such as those produced by LongRanger. We then retrieve barcode overlapping data from all samples for each region. These data are organized as a high dimensional matrix. The dimension of this matrix is then reduced using principal component analysis. Samples projected onto a two dimensional space formed by the first two principal components forms two or three clusters based on their genotype, representing the reference, alternative, or heterozygotic alleles. We developed a novel distance measure for hierarchical clustering and rotating the axes to find the optimal clustering results. We also developed an algorithm to decide whether the pattern of sample distribution is best fitted with one, two, or three genotypes. For each sample, we calculate its membership score for each genotype. We compared results produced by SVJAM with LongRanger and few methods that rely on PacBio or Oxford Nanopore data. In a comparison of SVJAM with SV detected using long-read sequencing data for the DBA/2J strain, we found that our results recovered many SVs missed by LongRanger. We also found many SVs called by LongRanger were assigned with an incorrect SV type. Our algorithm also consistently identified heterozygotic regions.

BIOM-39. METHYLATION AND MUTATION PROFILES IN MENINGIOMA CELL-DERIVED EXTRACELLULAR VESICLE DNA REFLECT EPIGENETIC AND GENOMIC ALTERATIONS IN ORIGINAL TUMORS

The majority of meningiomas are benign but approximately 20% of display an aggressive behavior, resulting in significant patient morbidity and mortality. Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering circulating EVs a potential biomarker source for non-invasive disease monitoring and for obtaining information on genetic and epigenetic alterations. We quantified EVs in plasma of 46 meningioma patients (n = 29 M1, 12 M2, 5 M3) by nanoparticle tracking analysis and detected significantly higher levels compared to age-matched healthy donors (n = 18). EV concentrations correlated with malignancy grade (p = 0.0049) and with the extent of peritumoral edema (p = 0.0031). Comparisons between paired pre- and postoperative samples revealed that EV levels counts dropped significantly the day after tumor resection and were reduced to normal levels after about one week. Completely resected patients (Simpson grade I) displayed a greater reduction of postoperative EV concentrations than incompletely resected patients. DNA methylation profiling was performed on EVs secreted by cultured meningioma cells, as well as matched cells and original tumors using 850k arrays (n = 7 M1, 5 M2, 3 M3). All EV samples were correctly identified as meningiomas by the Heidelberg classifier, and methylation subclasses were also correctly assigned in almost all cases. t-SNE analysis showed that EVs mapped in close proximity to their corresponding parental cells and tumor tissue. Tumor specific mutations and copy number variations were detected in EV-DNA with high accuracy. Differential quantitative proteomic analysis of EVs, cells and tumors identified shared proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids.

Repeatability of methylation measures using a QIAseq targeted methyl panel and comparison with the Illumina HumanMethylation450 assay

Objective In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). Results The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62–0.81), 0.59 (0.47–0.71) and 0.80 (0.73–0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39–0.68), 0.43 (0.31–0.56) and 0.70 (0.59–0.80), respectively. Bland–Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined.

The role of apoptosis gene methylation in the pathogenesis of breast and ovarian cancer

Актуальность. Рак молочной железы (РМЖ) и рак яичников (РЯ) наиболее часто диагностируемые типы рака у женщин, характеризующиеся своими клиническими особенностями, включая тяжёлое течение болезни и неблагоприятный прогноз. В России ежегодно умирают от этих видов опухолей более 29000 человек. Важную роль в патогенезе рака играет аберрантное метилирование CpG-островков промоторных областей в генах системы апоптоза. Ранее появились сообщения о гиперметилировании генов DAPK1, APAF1, BCL2, TP53 в некоторых видах опухолей. Однако, данные о роли метилирования этой группы генов в прогрессии РМЖ и РЯ представлены единичными сообщениями, а для генов BIM, BAX до настоящего момента до сих пор не представлены. Целью настоящей работы явилось исследование роли метилирования шести генов системы апоптоза, а именно, про-апоптозных генов APAF1, DAPK1, BIM, BAX, TP53, а также анти-апоптозного гена BCL2 в прогрессии РМЖ и РЯ. Методы. Образцы опухолей РМЖ и РЯ собраны и клинически охарактеризованы в НИИ клинической онкологии ФГБУ РОНЦ имени Н.Н. Блохина. Высокомолекулярную ДНК выделяли из ткани стандартным методом. Анализ уровня метилирования проводили с применением бисульфитной конверсии ДНК и количественной метилспецифичной ПЦР (МС-ПЦР) с детекцией в реальном времени. Для оценки значимости различий между исследуемыми группами применяли непараметрический критерий Манна-Уитни для независимых выборок. Результаты. Показано статистически значимое (р < 0,05) увеличение уровня метилирования генов BCL2, DAPK1 и BAX в образцах опухолей по сравнению с парной гистологически нормальной тканью яичников. При РМЖ наблюдали гиперметилирование четырёх про-апоптозных генов (DAPK1, APAF1, BIM, BAX) и, напротив, гипометилирование BCL2. Кроме того, нами выявлено, что уровень метилирования промоторных CpG-островков генов DAPK1, BIM, BAX, APAF1 статистически значимо коррелирует с клинической стадией (р < 0,001), с размером опухоли (р < 0,02), а для генов BIM, BAX - с метастазированием (р < 0,02) при РМЖ. Также нами было показано, что уровень метилирования гена BAX значимо коррелирует с клинической стадией; размером опухоли и с метастазированием при РЯ (р < 0,05). Заключение. Полученные нами данные показывают, какую роль метилирование исследованных генов апоптоза играет в возникновении и прогрессии РЯ и РМЖ и позволяют оценить их влияние на патофизиологический профиль опухолей яичников и молочной железы. Background. Breast cancer (BC) and ovarian cancer (OC) are the most commonly diagnosed types of cancer in women, which are characterized by severe course and unfavorable prognosis. In Russia, more than 29,000 people die from these types of tumors every year. Aberrant methylation of CpG islands located in the promoter regions of apoptosis genes play an important role in the pathogenesis of this disease. Previously, there were reports of hypermethylation of the DAPK1, APAF1, BCL2, and TP53 genes in some types of tumors. However, reports of the role of the gene methylation in the progression of BC and OC are scarce, and for the BIM and BAX genes, this information is absent. The aim of this study was to elucidate the role of methylation for six genes of the apoptosis system, namely, the pro-apoptotic genes APAF1, DAPK1, BIM, BAX, TP53, as well as the anti-apoptotic gene BCL2, in the progression of BC and OC. Methods. Samples of breast and ovarian tumors were collected and clinically characterized at the N.N. Blokhin Research Institute of Clinical Oncology. High molecular weight DNA was isolated from the tissue using standard methods. The methylation degree was assessed by bisulfite DNA conversion and real-time quantitative methylation-specific PCR (MS-PCR). Significance of differences between the study groups was determined with the nonparametric Mann-Whitney test for independent samples. Results. The degree of BCL2, DAPK1, and BAX gene methylation was significantly increased (p<0.05) in tumor samples compared to matched histologically normal ovarian tissue. Hypermethylation of four pro-apoptotic genes (DAPK1, APAF1, BIM, and BAX) and, in contrast, hypomethylation of BCL2 were observed in BC. In addition, in BC, the promoter CpG island methylation of DAPK1, BIM, BAX, APAF1 genes significantly correlated with the clinical stage (p < 0.001), and with the tumor size (p < 0.02) whereas for BIM and BAX genes, the methylation degree correlated with metastasis (p < 0.02). In OC, the methylation degree of the BAX gene significantly correlated with the clinical stage, with the tumor size, and with metastasis (p < 0.05). Conclusion. The results of this study showed the role of apoptosis gene methylation in the development and progression of BC and OC and also allowed evaluating their influence on the pathophysiological profile of ovarian and breast tumors.

Optimization of the “in-silico” mate-pair method improved contiguity and accuracy of genome assembly

A combination of next-generation sequencing technologies and mate-pair libraries of large insert sizes is used as a standard method to generate genome assemblies with high contiguity. The third-generation sequencing techniques also are used to improve the quality of assembled genomes. However, both mate-pair libraries and the third-generation libraries require high-molecular-weight DNA, making the use of these libraries inappropriate for samples with only degraded DNA. An in silico method that generates mate-pair libraries using a reference genome was devised for the task of assembling target genomes. Although the contiguity and completeness of assembled genomes were significantly improved by this method, a high level of errors manifested in the assembly, further to which the methods for using reference genomes were not optimized. Here, we tested different strategies for using reference genomes to generate in silico mate-pairs. The results showed that using a closely related reference genome from the same genus was more effective than using divergent references. Conservation of in silico mate-pairs by comparing two references and using those to guide genome assembly reduced the number of misassemblies (18.6% – 46.1%) and increased the contiguity of assembled genomes (9.7% – 70.7%), while maintaining gene completeness at a level that was either similar or marginally lower than that obtained via the current method. Finally, we compared the optimized method with another reference-guided assembler, RaGOO. We found that RaGOO produced longer scaffolds (17.8 Mbp vs 3.0 Mbp), but resulted in a much higher misassembly rate (85.68%) than our optimized in silico mate-pair method.

Something old, something new, a DNA preparation procedure for long read genomic sequencing

Extracting high molecular weight DNA is critical for successful long read DNA sequencing. Here we present a DNA preparation procedure combining CTAB DNA extraction protocol and a short read eliminator kit, which consistently produces high yields of high molecular weight DNA in the freshwater microcrustacean Daphnia. This method only requires common chemicals and equipment, providing researchers a viable alternative to DNA extraction kits that often require specialized tools.

Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle Tribolium and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.