A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

互联网2013-09-06

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The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes.

Key Words: bisulfite genomic sequencing - multiple restriction enzyme digestion - methylation

Introduction

The bisulfite genomic sequencing (BGS) protocol (1 , 2 ) is a method of choice for analyzing DNA methylation at the nucleotide level. Sodium bisulfite is used to convert unmethylated cytosine residues to uracil residues in single-stranded DNA. In particular, bisulfite conversion consists of three sequential chemical reactions: sulfonation of cytosine to cytosine-6-sulfonate, deamination to uracil-6-sulfonate, and desulfonation to uracil. However, since 5-methylcytosine residues are nonreactive, they remain intact. The bisulfite-converted DNA is then amplified with specific primers designed for converted DNA, and purified polymerase chain reaction (PCR) products, which are usually subcloned, are sequenced.

Bisulfite conversion is so powerful that it has been paired with numerous techniques other than traditional sequencing, including: methylation-specific PCR (3 ), combined bisulfite restriction enzyme analysis (4 ), methylation-sensitive single nucleotide primer extension (5 ), methylation-sensitive single-strand conformation analysis (6 ), MethyLight (7 ), oligonucleotide microarray methods (8 ), denaturing high-performance liquid chromatography with bisulfite genomic sequencing (9 ), pyrosequencing methylation analysis (10 ), and methylation-sensitive high-resolution melting-curve analysis (11 ), among others (see (12 ) for a review). In addition, many methylation analysis kits are also commercially available.

Unfortunately, high rates of DNA degradation and incomplete conversion reactions often lead to decreased efficiency of the assay. Many attempts have been made to minimize template degradation and/or maximize cytosine conversion (13 � 19 ), but overall, the bisulfite conversion protocol has remained unchanged, and no other high resolution or positive display methylation analysis protocol exists. As a result, the BGS protocol, as well as any technique paired with the bisulfite conversion reaction (and, hence, founded on the assumption that conversion is complete) often generate few or no informative results.

In our studies of the RARB2 P2 promoter (20 ), we found that incomplete conversion was an insurmountable challenge even after modifying the protocol in numerous ways. We, therefore, aimed to circumvent these issues altogether by depleting the PCR populations of products amplified from partially converted or unconverted DNA using a multiple restriction enzyme digestion (MRED) approach. We found that informative sequencings were increased ninefold using it. We believe that this method may easily be adapted for analyzing the detailed methylation status of other genes presenting incomplete cytosine to uracil conversion, and we provide guidelines for selecting the most appropriate restriction enzymes (REs).

Materials and Methods

Cell Culture and Genomic DNA Extraction

Cell-Line Provenance

Twenty-one cell lines were cultured. CALU-1, SK-MES, CACO-2, COLO-201, COLO-205, HCT-15, and LS-180 were obtained from the American Type Culture Collection (Rockville, MD). The CALU-1 daughter cell lines, C-19 and C-59, are RARB2 -transfectants that were established in our laboratory (21 ). MM-1 was also established in our laboratory (6 ). NCI-H23, NCI-H82, NCI-H125, NCI-H157, NCI-H520, and NCI-H596 were supplied by Dr. Adi Gazdar (NCI, NIH, Bethesda, MD). NBE-E₆ E₇ (22 ) was provided by Dr. Jean Viallet (Gemin X Biotechnologies Inc., Montreal, Québec). SW 1222 was given to us by Dr. Clifford Stanners (McGill University, Montreal, Québec). Qu-DB was provided by Dr. Barbara Campling (Queen’s University, Kingston, Ontario). T47D, MDA-MB-231 (MB-231), ZR-75B, and HS-578T were kindly provided by Dr. Morag Park (McGill University, Montreal, Québec).

Cell Culture

CALU-1, CACO-2, SW-1222, and LS-180 were grown in α-MEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Wisent Bioproducts, Saint-Jean-Baptiste de Rouville, Québec). NBE-E₆ E₇ was grown in keratinocyte-serum free medium (Invitrogen), supplemented with 50 µg/ml bovine pituitary extract, and 5 ng/ml recombinant human epidermal growth factor (Invitrogen). All other cells were grown in RPMI-1640 medium (Invitrogen) supplemented either with 5% (SK-MES, NCI-H23, NCI-H125, NCI-H520, Qu-DB, and HS-578T) or 10% FCS (NCI-H82, NCI-H157, MM-1, T47D, MDA-MB-231, ZR-75B, COLO-201, COLO-205, and HCT-15). Where indicated, cells were treated with 1 µM 5-azadeoxycytidine.

Genomic DNA Extraction

Genomic DNA was extracted using the standard phenol-chloroform technique followed by proteinase K treatment to ensure complete protein removal (23 ). DNA was then digested with the Pst I RE (New England BioLabs, Ipswich, MA) according to the supplier′s directives to shorten the fragment (2.95 kb) containing the target RARB2 P2 promoter sequence investigated (541 bp; Fig . 1 ), thereby reducing the possibility for regional double-strand formation (24 ). Pst I was the only RE available for the sequence under analysis.

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Fig. 1 RARB2 sequence under analysis. A Unconverted sequence; B converted sequence (all non-CpG-cytosines have been replaced with thymidines). The 541 bp sequence analyzed is comprised between oligonucleotides JP5 and JP7. CG = CpG dinucleotide under investigation (n = 22); T = non-CpG-cytosine converted to thymidine following bisulfite treatment (n = 82); boxes promoter elements; gray-shaded sequences RE sites (please note that these sites are absent in the converted sequence); +1 transcription start site. Direct repeats of the RARE are indicated. The oligonucleotide sequences are underlined and are designed for converted DNA.