LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis

Background DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs. Results We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis. Conclusions In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.

Standardization of DNA amount for bisulfite conversion for analyzing the methylation status of LINE-1 in lung cancer

Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300–368 as LINE-1.1 and 368–460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.

DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps

We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Forster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.

Methylation of MMP2 and MMP9 in patients with localized and generalized forms of Ewing's sarcoma.

e23503 Background: MMPs play a critical role in tumor growth and progression, metastatic development, angiogenesis, and tumor invasion. Epigenetic regulation of MMP2 and MMP9 levels is often disrupted in cancer and is considered as potential biomarkers. In this study, we tried to assess the methylation of the MMP2 and MMP9 genes in patients with localized and generalized forms of Ewing's sarcoma (ES). Methods: As a material used DNA from FFPE of primary tumor of 20 patients with localized forms ES and 20 patients with generalized forms ES. After bisulfite conversion of total DNA and PCR, the level of CpG methylation was assessed by sequencing on a Genetic Analyzer Applied Biosystems 3500. Results: The analysis showed the presence of hypomethylation of the MMP2 in the group with generalized forms (100%) 1.25 times more often than in the group with localized forms (80%), and hypermethylation was observed only in the group with localized forms–20% of cases (χ2 = 4.234, p = 0.040). On the other hand, hypermethylation was 1.9 times more common in the group with localized forms of ES (90%) (χ2 = 8.313, p = 0.004). Differences in the methylation status of the MMP2 and MMP9 indicate that the presence of hypomethylation at two loci increases the risk of developing generalized ES by 22 times (OR = 22.2, p < 0.05; CI95% 2.460-20.769). Conclusions: Specific methylation of the matrix metalloproteinases genes MMP2 and MMP9 identified in the groups of localized and generalized Ewing's sarcoma. At the same time, the group with generalized forms of ES was characterized by gene hypomethylation.

Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion

Bisulfite (BS) conversion, which includes a series of chemical reactions using bisulfite, is a prerequisite to most DNA methylation analysis methods, and thus is an essential step in the associated research process. Unfortunately, BS conversion leads to the degradation or loss of DNA, which can hinder further downstream analysis. In addition, it is well known that incomplete BS conversion is crucial, as it causes an exaggeration of the DNA methylation level, which can adversely affect the results. Therefore, there have been many attempts to measure three key features of BS conversion: BS conversion efficiency, recovery, and degradation level. In this study, a multiplex quantitative real-time PCR system named BisQuE was suggested to simultaneously analyze three important aspects of the conversion step. By adopting cytosine-free PCR primers for two differently sized multicopy regions, the short amplicon and long amplicon were obtained from both the genomic and BS-converted DNA, thus enabling the obtaining of reliable and sensitive results and the calculation of the degradation level of the conversion step. Also, probes for detecting converted/unconverted templates and C-T indicators for inducing the formula were included in this assay to quantify BS-converted DNA in order to compute the conversion efficiency and recovery. Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE. The conversion efficiency, degradation levels, as well as recovery rates of the kits were investigated. A total of 99.61–99.90% conversion efficiency was perceived for five of the kits, while the NEBNext kit showed about 94%. The lowest degradation level was shown by the NEBNext kit, whereas the other kits were quite similar. The recovery rates of the kits were found to be within the range of 18–50%. A Qubit assay was also used to compare the recovery rate of BisQuE.

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional Bayesian approaches

Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional tools, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and for resolving the interaction between genetic and epigenetic effects when elucidating the DNA methylome. The confounding effect of bisulfite conversion can be resolved by observing differences in allele counts on a per-strand basis. Herein, we present a computational pre-processing approach for adapting such data, thus enabling downstream analysis in this way using conventional variant calling software such as GATK or Freebayes.

Μεθυλίωση και απενεργοποίηση των γονιδίων RASSF1A και RAR-β2 στην καρκινογένεση του μαστού

Υπόβαθρο: Η DNA υπερμεθυλίωση γονιδιακών υποκινητών είναι αρκετά συχνό φαινόμενο στον Καρκίνο του Μαστού, με τις περισσότερες μελέτες μέχρι σήμερα που βασίζονται σε ιστολογικό υλικό από βιοψίες μαστού να καταδεικνύουν συχνή μεθυλίωση σε ογκοκατασταλτικά γονίδια σε κακοήθεις ιστούς σε σχέση με φυσιολογικούς ιστούς. Σκοπός αυτής της διατριβής ήταν ο προσδιορισμός της συχνότητάς μεθυλίωσης των υποκινητών των γονιδίων RAR-β2 & RASSF1A σε κυτταρολογικό υλικό από FNAB μαστού με σκοπό να διερευνηθεί εάν η κατάσταση μεθυλίωσης αυτών των 2 γονιδίων μπορεί να χρησιμοποιηθεί ως βιοδεικτής για την ανίχνευση περιστατικών κακοήθειας μαστού σε ελληνικό πληθυσμό. Μεθοδολογία: Σε υλικό FNAB μαστού από 104 ασθενείς πραγματοποιήθηκε κυτταρολογική εξέταση και επιγενετική ανάλυση μεθυλίωσης. DNA απομονώθηκε από τα κλινικά δείγματα και υφίστατο τροποποίηση παρουσία όξινου θειώδους νατρίου (bisulfite conversion). Σχεδιάστηκε και εφαρμόστηκε ένα ειδικό πρωτόκολλο methylation-specific PCR (MSP) και τα τελικά MSP προϊόνταδια χωρίστηκαν με ηλεκτροφόρηση σε 2% πηκτώματα αγαρόζης. Αποτελέσματα: RASSF1A υπερμεθυλίωση παρατηρήθηκε σε 78 (75%) και RAR-β2 υπερμεθυλίωση σε 64 (61,6%) από τα συνολικά 104 περιστατικά. 84% και 78% από τα περιστατικά C5 (n=50) ήταν μεθυλιωμένα στα γονίδια RASSF1A και RAR-β2 αντιστοίχως. Μεθυλίωση στα γονίδια RASSF1A και RAR-β2 ανιχνεύτηκε επίσης σε 88,3% και 76,5% στα περιστατικά C4 (n=17) και σε 57,2% των περιστατικών C3 (n=14). Ο σχετικός κίνδυνος για κακοήθεια μαστού ήταν 4,545 σε ασθενείς με υπερμεθυλίωση του υποκινητή του γονιδίουRASSF1A και 9,167 σε ασθενείς με υπερμεθυλίωση του υποκινητή του γονιδίου RAR-β2 υποδηλώνοντας τη θετική συσχέτιση των 2 αυτών μεθυλιωμένων δεικτών με την εμφάνιση κακοήθειας μαστού. Συμπέρασμα: Για τη βελτιστοποίηση της ευαισθησίας και ειδικότητας του διγονιδιακού πάνελ μεθυλίωσης που διερευνάται στα πλαίσια αυτής της διατριβής, απαιτείται εμπλουτισμός του με περισσότερα σχετιζόμενα με τον καρκίνο του μαστού ογκοκατασταλτικά γονίδια και αξιολόγηση του σε μεγαλύτερο δείγμα μελέτης με σκοπό τα επιγενετικά δεδομένα μεθυλίωσης που θα συγκεντρωθούν από τις αναρροφήσεις FNAB μαστού να παρέχουν στους κλινικούς ιατρούς πολύτιμα εργαλεία στην διαχείριση περιστατικών κακοήθειας μαστού στην Ελλάδα.