the widely used 450k dna methylation array ( infinium humanmethylation450 beadchip ) includes 485,577 cytosine positions in human dna distributed among all 22 autosomal and 1 sex chromosome pair . when correlated to rna transcripts , 74.4% of cpgs correspond to coding messenger rna genes , 0.85% are associated to noncoding rnas ( mirnas and long noncoding rnas ) and 24.6% sites do not possess an associated transcript . analysis of genomic distribution shows 30.9% of the sites are in cpg islands , 23% in cpg shores ( sequences 2 kb upstream and downstream from cpg island ) , 9.7% in cpg shelves ( sequences 2 kb upstream and downstream from shore regions ) and 36.3% are located outside these regions (  open sea ) . following a functional classification , 41% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp or 1500 bp upstream of transcription start sites , exon 1 and in 5utrs ) , 3.2% correspond to 3utrs , 30.9% to gene bodies and 24.6% to intergenic regions . the recently developed methylationepic beadchip infinium microarray interrogates the methylation status of 853,307 cpg sites . there are 439,562 cpgs from the 482,421 cpgs included in the 450k microarray ( 91.1% ) , that are also present in the 850k dna methylation microarray . the complete list of cpgs shared by both microarrays is shown in supplementary table 1 . the 42,076 cpg sites ( 8.9% ) that were included in the 450k microarray that are not present in the 850k microarray are listed in supplementary table 2 . most importantly , the methylationepic beadchip infinium includes 413,745 new cpg sites not included in the 450k microarray that interrogate the dna methylation status of other sequences of the genome , which are shown in supplementary table 3 . the methylationepic beadchip infinium also interrogates 2880 cng ( c stands for cytosine ; n stands for any nucleotide ; g stands for guanine ) sites that are present in the 450k dna methylation microarray ( supplementary table 4 ) , while 211 cng sites from the 450k microarray are not included in the 850k platform . the 850k microarray also includes 59 snp sites that are used for quality control processes ( supplementary table 5 ) . the degree of cpg site overlap between the 450k and the 850k dna methylation microarrays is summarized in figure 1 . the 413,745 cpg newly added positions in the 850k microarray are localized among all 22 autosomal chromosomes in addition to the x and y sex chromosomes ( figure 2a ) . chromosome 1 harbors the most positions ( 39,087 , 9.4% ) and chromosome y the fewest ( 179 , 0.04% ) . according to the manufacturer 's technical notes , most ( 95.1% ) of them used the infinium design ii , while only 4.9% used infinium design i ( figure 2b ) . following a functional classification , 25.8% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 5.4% ] or 1500 bp [ 10.6% ] upstream of transcription start sites , exon 1 [ 0.4% ] and in 5utrs [ 9.4% ] ) , 0.9% correspond to 3utrs , 40% to gene bodies and 33.3% to intergenic regions ( figure 2c ) . relative to cpg context , 5.4% of the sites are in cpg islands , 11.4% in cpg shores , 5% in cpg shelves and , most importantly , the vast majority ( 78.2% ) are located outside these regions (  open sea ) ( figure 2d ) . the observation that the newly included cpgs in the 850k dna methylation microarray are mainly located in sequences with poor cpg content , in addition to its intergenic location ( 33.3% ) , fits with the concept that these cpg sites were incorporated from the human enhancer regions provided by the encode   and fantom5   projects . for example , 4.9% of all the cpg sites are derived from a fantom5 defined enhancer ( figure 2e ) . with respect to encode regulatory elements , 19.3% of the cpgs are located within a known transcription factor binding site identified by chip - seq ( chromatin immunoprecipitation and sequencing ) ( figure 2f ) , 14.6% are in open chromatin regions defined by faire - seq ( formaldehyde - assisted isolation of regulatory elements sequencing ) ( figure 2 g ) and 53.6% of the cpgs correspond to dnase hypersensitive regions identified by dnase - seq ( figure 2h ) . the genomic and functional context of all the 853,307 cpgs included in the methylationepic beadchip infinium assay , including those exported from the 450k microarray ( 439,562 ) with those newly incorporated in the 850k microarray ( 413,745 cpgs ) was analyzed next in whole . all 22 autosomal chromosomes in addition to the x and y sex chromosomes are present , with chromosome 1 harboring the most positions ( 9.5% ) and chromosome y the fewest ( 0.1% ) ( figure 3a ) . considering infinium design , most cpgs used design ii ( 83.8% ) , whereas 16.2% used design i ( figure 3b ) . to quantify the possible false cpg methylation calling due to the presence of a snp , we analyzed how many 850k cpg probes have an snp within their last ten bases and with a minor allele frequency ( maf ) 5% according to the 1000 human genome project database . we observed that 8.8% of the cpg sites had an associated snp with the described features ( supplementary table 6 ) . according to associated rna transcripts , 623,483 cpgs ( 73.1% ) correspond to classic coding messenger rna genes , while 21,446 ( 2.5% ) are linked to noncoding rnas ( 9961 for mirnas and 11,485 for long noncoding rnas ) . for 229,824 ( 26.9% ) sites addressing the issue of functional classification , 35.2% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 9.4% ] or 1500 bp [ 13.9% ] upstream of transcription start sites , exon 1 [ 1.2% ] and in 5utrs [ 10.7% ] ) , 1.9% correspond to 3utrs , 35.9% to gene bodies and 26.9% to intergenic regions ( figure 3c ) . as a function of cpg landmarks , 17.8% of the sites are in cpg islands , 16.9% in cpg shores and 8.8% in cpg shelves , but most of the cpg sites ( 56.5% ) are not in any of these sequences (  open sea ) ( figure 3d ) . if we combine the two classifications described above , we can determine that for the 300,554 cpg sites located in proximal promoters , 102,892 cpgs ( 34.2% ) are in cpg islands , 84,491 ( 28.1% ) in cpg shores and 18,393 ( 6.1% ) in cpg shelves , while 94,778 ( 31.5% ) are in other regions of the genome without any enrichment of cpg content ( open sea ) ( figure 3 ) . related to regulatory regions , 3.2% of the cpg sites are within fantom5 described enhancer regions ( figure 3e ) , covering 36.3% of the described fantom5 enhancers with at least one cpg site . the included fantom5 enhancers are within a cpg island , shelf or shore in 26.6% of cases , whereas the remaining 73.4% are outside these cpg sequence categories . in addition , 15.6% of the cpgs are located within a known transcription factor binding site ( figure 3f ) , 13.8% are in open chromatin regions ( figure 3 g ) ( covering 38.7% of overlapped open chromatin regions annotated in the encode project ) and 57.4% correspond to cpgs within dnase i hypersensitive regions ( figure 3h ) . other detailed information about the cpgs included in the 850k array can be found at the manufacturer website . to prove the utility and reliability of the infinium methylationepic beadchip for the analysis of dna methylation first , we provided a technical validation of the 850k dna methylation microarray data by studying a primary renal tumor ( rcc9 ) , and comparing the obtained results from the well established and fully standardized 450k platform . we observed that dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected at each cpg site using the 450k microarray ( pearson correlation coefficient r = 0.992 ; p  2.22e-16 ) ( figure 4a ) . we also performed a replication experiment by hybridizing twice the same sample ( normal colon nc22a ) to the 850k microarray . we found that the methylation levels detected at cpg sites derived from each experiment were highly correlated and completely interchangeable ( pearson correlation coefficient r = 0.997 ; p  2.22e-16 ) ( figure 4b ) . given the excellent performance of the 450k microarray for formalin - fixed paraffin - embedded ( ffpe ) samples , we wondered about the reliability of the 850k microarray to determine the dna methylation patterns in this type of archival material . to address this issue , we hybridized to the platform the same dna sample obtained from fresh tissue ( primary renal tumor rcc9 ) or derived after the formalin - fixing paraffin - embedding procedure ( rcc9-ffpe ) . we observed that the methylation levels detected at each cpg site derived from each sample were highly correlated ( pearson correlation coefficient r = 0.994 ; p  2.22e-16 ) ( figure 4c ) . cellular heterogeneity is a potential limitation of this part of the study , but as the ffpe section was close to the fresh - frozen section significant cellular heterogeneity is not expected . we also sought to demonstrate that the newly developed 850k dna methylation microarray was also valuable for the study of 5-hydroxymethylcytosine patterns , as has been shown for the 450k platform . to accomplish this aim , we carried out oxidation of dna purified from sorted neurons ( n229 ) , a tissue particularly enriched for 5-hydroxymethylcytosine , using the truemethyl array kit ( cambridge epigenetix , cambridge , uk ) where the final eluted fraction followed the methylationepic processing according to the kit instructions for infinium arrays . using this approach , we were able to determine the 5- hydroxymethylcytosine patterns in the sorted neurons ( figure 4d & e ) . the genomic distribution of 5-hydroxymethylcytosine , their functional annotation and genomic feature enrichment are shown in supplementary figure 1 . finally , we assessed the ability of the 850k microarray to identify differential methylated cpg sites to allow for the study of various biological and biomedical phenomena . for this purpose , we used unsupervised dna clustering on the 850k methylation data from tissues of varying developmental origin , such as colon mucosa ( nc22a ) and neurons ( n229 ) , and were able to discriminate the two normal tissues based on their methylation profiles ( figure 5 ) . overall , 1468 cpg sites displayed differential methylation levels with delta beta values higher than 66% between these two cell types ( figure 5 & supplementary table 7 ) . if we lowered the threshold to a 33% delta value , 73,774 cpg sites are distinct between these normal tissues ( supplementary table 8) . for these differentially methylated cpgs , 6.2% and 12.6% were located in fantom5 enhancers and open chromatin regions defined by encode , respectively . most importantly , 40.5% ( at delta beta value of 66% ) ( figure 5 & supplementary table 9 ) and 55.6% ( with delta beta value of 33% ) ( supplementary table 10 ) of these differentially methylated sites corresponded to the newly incorporated cpgs in the 850k , a fraction that due to its enrichment in enhancer sequences could represent a powerful tool to dissect cellular and tissue lineages among different experimental conditions and clinical entities . the complete 850k and 450k data from all the described samples are available for download from ncbi geo . the widely used 450k dna methylation array ( infinium humanmethylation450 beadchip ) includes 485,577 cytosine positions in human dna distributed among all 22 autosomal and 1 sex chromosome pair . when correlated to rna transcripts , 74.4% of cpgs correspond to coding messenger rna genes , 0.85% are associated to noncoding rnas ( mirnas and long noncoding rnas ) and 24.6% sites do not possess an associated transcript . analysis of genomic distribution shows 30.9% of the sites are in cpg islands , 23% in cpg shores ( sequences 2 kb upstream and downstream from cpg island ) , 9.7% in cpg shelves ( sequences 2 kb upstream and downstream from shore regions ) and 36.3% are located outside these regions (  open sea ) . following a functional classification , 41% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp or 1500 bp upstream of transcription start sites , exon 1 and in 5utrs ) , 3.2% correspond to 3utrs , 30.9% to gene bodies and 24.6% to intergenic regions . the recently developed methylationepic beadchip infinium microarray interrogates the methylation status of 853,307 cpg sites . there are 439,562 cpgs from the 482,421 cpgs included in the 450k microarray ( 91.1% ) , that are also present in the 850k dna methylation microarray . the complete list of cpgs shared by both microarrays is shown in supplementary table 1 . the 42,076 cpg sites ( 8.9% ) that were included in the 450k microarray that are not present in the 850k microarray are listed in supplementary table 2 . most importantly , the methylationepic beadchip infinium includes 413,745 new cpg sites not included in the 450k microarray that interrogate the dna methylation status of other sequences of the genome , which are shown in supplementary table 3 . the methylationepic beadchip infinium also interrogates 2880 cng ( c stands for cytosine ; n stands for any nucleotide ; g stands for guanine ) sites that are present in the 450k dna methylation microarray ( supplementary table 4 ) , while 211 cng sites from the 450k microarray are not included in the 850k platform . the 850k microarray also includes 59 snp sites that are used for quality control processes ( supplementary table 5 ) . the degree of cpg site overlap between the 450k and the 850k dna methylation microarrays is summarized in figure 1 . the 413,745 cpg newly added positions in the 850k microarray are localized among all 22 autosomal chromosomes in addition to the x and y sex chromosomes ( figure 2a ) . chromosome 1 harbors the most positions ( 39,087 , 9.4% ) and chromosome y the fewest ( 179 , 0.04% ) . according to the manufacturer 's technical notes , most ( 95.1% ) of them used the infinium design ii , while only 4.9% used infinium design i ( figure 2b ) . following a functional classification , 25.8% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 5.4% ] or 1500 bp [ 10.6% ] upstream of transcription start sites , exon 1 [ 0.4% ] and in 5utrs [ 9.4% ] ) , 0.9% correspond to 3utrs , 40% to gene bodies and 33.3% to intergenic regions ( figure 2c ) . relative to cpg context , 5.4% of the sites are in cpg islands , 11.4% in cpg shores , 5% in cpg shelves and , most importantly , the vast majority ( 78.2% ) are located outside these regions (  open sea ) ( figure 2d ) . the observation that the newly included cpgs in the 850k dna methylation microarray are mainly located in sequences with poor cpg content , in addition to its intergenic location ( 33.3% ) , fits with the concept that these cpg sites were incorporated from the human enhancer regions provided by the encode   and fantom5   projects . for example , 4.9% of all the cpg sites are derived from a fantom5 defined enhancer ( figure 2e ) . with respect to encode regulatory elements , 19.3% of the cpgs are located within a known transcription factor binding site identified by chip - seq ( chromatin immunoprecipitation and sequencing ) ( figure 2f ) , 14.6% are in open chromatin regions defined by faire - seq ( formaldehyde - assisted isolation of regulatory elements sequencing ) ( figure 2 g ) and 53.6% of the cpgs correspond to dnase hypersensitive regions identified by dnase - seq ( figure 2h ) . the genomic and functional context of all the 853,307 cpgs included in the methylationepic beadchip infinium assay , including those exported from the 450k microarray ( 439,562 ) with those newly incorporated in the 850k microarray ( 413,745 cpgs ) was analyzed next in whole . all 22 autosomal chromosomes in addition to the x and y sex chromosomes are present , with chromosome 1 harboring the most positions ( 9.5% ) and chromosome y the fewest ( 0.1% ) ( figure 3a ) . considering infinium design , most cpgs used design ii ( 83.8% ) , whereas 16.2% used design i ( figure 3b ) . to quantify the possible false cpg methylation calling due to the presence of a snp , we analyzed how many 850k cpg probes have an snp within their last ten bases and with a minor allele frequency ( maf ) 5% according to the 1000 human genome project database . we observed that 8.8% of the cpg sites had an associated snp with the described features ( supplementary table 6 ) . according to associated rna transcripts , 623,483 cpgs ( 73.1% ) correspond to classic coding messenger rna genes , while 21,446 ( 2.5% ) are linked to noncoding rnas ( 9961 for mirnas and 11,485 for long noncoding rnas ) . for 229,824 ( 26.9% ) sites addressing the issue of functional classification , 35.2% of the cpgs are located in proximal promoters ( cpg sites located within 200 bp [ 9.4% ] or 1500 bp [ 13.9% ] upstream of transcription start sites , exon 1 [ 1.2% ] and in 5utrs [ 10.7% ] ) , 1.9% correspond to 3utrs , 35.9% to gene bodies and 26.9% to intergenic regions ( figure 3c ) . as a function of cpg landmarks , 17.8% of the sites are in cpg islands , 16.9% in cpg shores and 8.8% in cpg shelves , but most of the cpg sites ( 56.5% ) are not in any of these sequences (  open sea ) ( figure 3d ) . if we combine the two classifications described above , we can determine that for the 300,554 cpg sites located in proximal promoters , 102,892 cpgs ( 34.2% ) are in cpg islands , 84,491 ( 28.1% ) in cpg shores and 18,393 ( 6.1% ) in cpg shelves , while 94,778 ( 31.5% ) are in other regions of the genome without any enrichment of cpg content ( open sea ) ( figure 3 ) . related to regulatory regions , 3.2% of the cpg sites are within fantom5 described enhancer regions ( figure 3e ) , covering 36.3% of the described fantom5 enhancers with at least one cpg site . the included fantom5 enhancers are within a cpg island , shelf or shore in 26.6% of cases , whereas the remaining 73.4% are outside these cpg sequence categories . in addition , 15.6% of the cpgs are located within a known transcription factor binding site ( figure 3f ) , 13.8% are in open chromatin regions ( figure 3 g ) ( covering 38.7% of overlapped open chromatin regions annotated in the encode project ) and 57.4% correspond to cpgs within dnase i hypersensitive regions ( figure 3h ) . other detailed information about the cpgs included in the 850k array can be found at the manufacturer website . to prove the utility and reliability of the infinium methylationepic beadchip for the analysis of dna methylation , we performed five different tests . first , we provided a technical validation of the 850k dna methylation microarray data by studying a primary renal tumor ( rcc9 ) , and comparing the obtained results from the well established and fully standardized 450k platform . we observed that dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected at each cpg site using the 450k microarray ( pearson correlation coefficient r = 0.992 ; p  2.22e-16 ) ( figure 4a ) . we also performed a replication experiment by hybridizing twice the same sample ( normal colon nc22a ) to the 850k microarray . we found that the methylation levels detected at cpg sites derived from each experiment were highly correlated and completely interchangeable ( pearson correlation coefficient r = 0.997 ; p  2.22e-16 ) ( figure 4b ) . given the excellent performance of the 450k microarray for formalin - fixed paraffin - embedded ( ffpe ) samples , we wondered about the reliability of the 850k microarray to determine the dna methylation patterns in this type of archival material . to address this issue , we hybridized to the platform the same dna sample obtained from fresh tissue ( primary renal tumor rcc9 ) or derived after the formalin - fixing paraffin - embedding procedure ( rcc9-ffpe ) . we observed that the methylation levels detected at each cpg site derived from each sample were highly correlated ( pearson correlation coefficient r = 0.994 ; p  2.22e-16 ) ( figure 4c ) . cellular heterogeneity is a potential limitation of this part of the study , but as the ffpe section was close to the fresh - frozen section significant cellular heterogeneity is not expected . we also sought to demonstrate that the newly developed 850k dna methylation microarray was also valuable for the study of 5-hydroxymethylcytosine patterns , as has been shown for the 450k platform . to accomplish this aim , we carried out oxidation of dna purified from sorted neurons ( n229 ) , a tissue particularly enriched for 5-hydroxymethylcytosine , using the truemethyl array kit ( cambridge epigenetix , cambridge , uk ) where the final eluted fraction followed the methylationepic processing according to the kit instructions for infinium arrays . using this approach , we were able to determine the 5- hydroxymethylcytosine patterns in the sorted neurons ( figure 4d & e ) . the genomic distribution of 5-hydroxymethylcytosine , their functional annotation and genomic feature enrichment are shown in supplementary figure 1 . finally , we assessed the ability of the 850k microarray to identify differential methylated cpg sites to allow for the study of various biological and biomedical phenomena . for this purpose , we used unsupervised dna clustering on the 850k methylation data from tissues of varying developmental origin , such as colon mucosa ( nc22a ) and neurons ( n229 ) , and were able to discriminate the two normal tissues based on their methylation profiles ( figure 5 ) . overall , 1468 cpg sites displayed differential methylation levels with delta beta values higher than 66% between these two cell types ( figure 5 & supplementary table 7 ) . if we lowered the threshold to a 33% delta value , 73,774 cpg sites are distinct between these normal tissues ( supplementary table 8) . for these differentially methylated cpgs , 6.2% and 12.6% were located in fantom5 enhancers and open chromatin regions defined by encode , respectively . most importantly , 40.5% ( at delta beta value of 66% ) ( figure 5 & supplementary table 9 ) and 55.6% ( with delta beta value of 33% ) ( supplementary table 10 ) of these differentially methylated sites corresponded to the newly incorporated cpgs in the 850k , a fraction that due to its enrichment in enhancer sequences could represent a powerful tool to dissect cellular and tissue lineages among different experimental conditions and clinical entities . the complete 850k and 450k data from all the described samples are available for download from ncbi geo . samples included in the study were all from human origin including a primary normal colon ( nc22a ) that was hybridized twice to validate technical reproducibility , primary sorted neurons ( n229 ) , as well as a biopsy of a renal cancer ( rcc9 ) which was divided in two pieces , one was stored as fresh - frozen ( ff ) tissue embedded in optimal cutting temperature ( oct ) compound stored at -80c , while the other piece was formalin - fixed for 8 h followed by paraffin - embedding ( ffpe ) and stored at room temperature ( rcc9-ffpe ) . all samples were extracted using column - based dna extraction method ( ffpe , e.z.n.a . , ga , usa ; ff , dneasy blood & tissue kit ; qiagen , hilden , germany ) following manufacturer 's instructions . all dna samples were treated with rnasea for 1 h at 45c , quantified by the fluorometric method ( quant - it picogreen dsdna assay , life technologies , ca , usa ) , and assessed for purity by nanodrop ( thermo scientific , ma , usa ) 260/280 and 260/230 ratio measurements . all dnas from ffpe blocks were checked for their suitability for ffpe restoration , as indicated by the infinium hd ffpe qc assay ( illumina inc . ) , by performing a quantitative pcr with 2 ng of ffpe dna . cq was calculated by subtracting the average value of cq of the interrogated sample from the cq value of a standard provided by the manufacturer . all ffpe samples had a cq < 5 , which is the recommended threshold for suitability for ffpe restoration . site - specific oxidation was carried out on 1 g of gdna of the sorted neurons in order to convert 5-hydroxymethyl cytosines ( 5-hmc ) into its formyl derivative 5-formylcytosine ( 5-fc )   using truemethyl array kit ( cambridge epigenetix ) following manufacturer 's instructions . both aliquots were denatured , and 1 l of oxidant solution was added to one of the aliquots ( ox - bs ) while the other one ( bs ) underwent a mock - up oxidation process by adding 1 l of ultrapure h20 . after incubating both aliquots at 40c for 30 min , and centrifuging to eliminate precipitates , a bisulfite conversion reaction was performed with the supernatant using supplied reagents . desulfonation and cleanup process were applied using provided reagents before eluting the dna with 10 l of elution buffer . the eluate was then transferred directly to methylationepic processing following kit instructions for infinium arrays . three hundred nanograms of ffpe dna , or 600 ng of ff dna were randomly distributed on a 96-well plate , and processed using the ez-96 dna methylation kit ( zymo research corp . , bisulfite - converted dna ( bs - dna ) from ffpe samples was processed as previously described . an 1-h reaction at 37c was then performed with ppr ( primer pre restore ) reagent and amr ( amplification mix restore reagent ) reagents supplied by the kit manufacturer , in which dna repair is accomplished . dna was cleaned with a zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 13 l of erb . cleaned dna was then denatured for 2 min at 95c , followed by ligation incubation at 37c for 1 h with rst and cmm reagents . the resulting material was cleaned with zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 10 l of dih20 . methylationepic beadarray shares the infinium hd chemistry assay ( illumina inc . ) used to interrogated the cytosine markers with humanmethylation450 beadchip . thus , the applicable protocol for methylationepic is the same as for humanmethylation450 , which is the infinium hd methylation assay protocol . eight microliter of restored ffpe bs - dna , 4 l of ff bs - dna or 7 l of samples that underwent oxidation protocol were used to process them following the illumina infinium hd methylation assay protocol , as previously described . the only difference was that the hybridization volume of processed sample used to load the microarray was 26 l instead of the 15 l used in the case of humanmethylation450 . the resulting raw data ( idats ) were normalized ( control normalization ) and background corrected using the methylation module ( 1.9.0 ) available on genomestudio ( v2011.1 ) software . the complete 850k and 450k data from all the described samples are available for download from ncbi geo . cpg markers present on methylationepic were classified based on its chromosome location , the infinium chemistry used to interrogate the marker ( infinium i , infinium ii ) and the feature category gene region as per ucsc annotation ( tss200 , tss1500 , 5utr , 1st exon , body , 3utr ) . related to this last classification , categories included tss200 as the region between 0 and 200 bases upstream from the transcriptional start site ( tss ) ; tss1500 category , 2001500 bases upstream tss ; 5utr included the region between the tss and the start site ( atg ) ; cpgs within the first exon of a gene were considered as 1st exon category ; cpgs downstream the first exon including intronic regions until the stop codon , were classified as gene body ; cpgs located downstream the stop codon until the poly a signal were considered as 3utr ; and cpgs that were not classified in any of the previous categories were annotated as intergenic . when multiple genes or tss were associated with a cpg site , category prioritization was applied following a 5-prime to 3-prime criteria ( tss200 > tss1500 > 5utr > 1st exon > body > additional criteria included the location of the marker relative to the cpg island ( open sea , island , shore , shelf ) , fantom 5-associated enhancer regions and regulatory regions described on encode project   such as transcription binding site sequences , open chromatin regions and digital dnase i hypersensitivity clusters . samples included in the study were all from human origin including a primary normal colon ( nc22a ) that was hybridized twice to validate technical reproducibility , primary sorted neurons ( n229 ) , as well as a biopsy of a renal cancer ( rcc9 ) which was divided in two pieces , one was stored as fresh - frozen ( ff ) tissue embedded in optimal cutting temperature ( oct ) compound stored at -80c , while the other piece was formalin - fixed for 8 h followed by paraffin - embedding ( ffpe ) and stored at room temperature ( rcc9-ffpe ) . all samples were extracted using column - based dna extraction method ( ffpe , e.z.n.a . , ga , usa ; ff , dneasy blood & tissue kit ; qiagen , hilden , germany ) following manufacturer 's instructions . all dna samples were treated with rnasea for 1 h at 45c , quantified by the fluorometric method ( quant - it picogreen dsdna assay , life technologies , ca , usa ) , and assessed for purity by nanodrop ( thermo scientific , ma , usa ) 260/280 and 260/230 ratio measurements . all dnas from ffpe blocks were checked for their suitability for ffpe restoration , as indicated by the infinium hd ffpe qc assay ( illumina inc . ) , by performing a quantitative pcr with 2 ng of ffpe dna . cq was calculated by subtracting the average value of cq of the interrogated sample from the cq value of a standard provided by the manufacturer . all ffpe samples had a cq < 5 , which is the recommended threshold for suitability for ffpe restoration . site - specific oxidation was carried out on 1 g of gdna of the sorted neurons in order to convert 5-hydroxymethyl cytosines ( 5-hmc ) into its formyl derivative 5-formylcytosine ( 5-fc )   using truemethyl array kit ( cambridge epigenetix ) following manufacturer 's instructions . both aliquots were denatured , and 1 l of oxidant solution was added to one of the aliquots ( ox - bs ) while the other one ( bs ) underwent a mock - up oxidation process by adding 1 l of ultrapure h20 . after incubating both aliquots at 40c for 30 min , and centrifuging to eliminate precipitates , a bisulfite conversion reaction was performed with the supernatant using supplied reagents . desulfonation and cleanup process were applied using provided reagents before eluting the dna with 10 l of elution buffer . the eluate was then transferred directly to methylationepic processing following kit instructions for infinium arrays . three hundred nanograms of ffpe dna , or 600 ng of ff dna were randomly distributed on a 96-well plate , and processed using the ez-96 dna methylation kit ( zymo research corp . , ca , usa ) following the manufacturer 's recommendations for infinium assays . bisulfite - converted dna ( bs - dna ) from ffpe samples was processed as previously described . an 1-h reaction at 37c was then performed with ppr ( primer pre restore ) reagent and amr ( amplification mix restore reagent ) reagents supplied by the kit manufacturer , in which dna repair is accomplished . dna was cleaned with a zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 13 l of erb . cleaned dna was then denatured for 2 min at 95c , followed by ligation incubation at 37c for 1 h with rst and cmm reagents . the resulting material was cleaned with zr-96 dna clean & concentrator-5 kit ( zymo research corp . ) and eluted in 10 l of dih20 . methylationepic beadarray shares the infinium hd chemistry assay ( illumina inc . ) used to interrogated the cytosine markers with humanmethylation450 beadchip . thus , the applicable protocol for methylationepic is the same as for humanmethylation450 , which is the infinium hd methylation assay protocol . eight microliter of restored ffpe bs - dna , 4 l of ff bs - dna or 7 l of samples that underwent oxidation protocol were used to process them following the illumina infinium hd methylation assay protocol , as previously described . the only difference was that the hybridization volume of processed sample used to load the microarray was 26 l instead of the 15 l used in the case of humanmethylation450 . the resulting raw data ( idats ) were normalized ( control normalization ) and background corrected using the methylation module ( 1.9.0 ) available on genomestudio ( v2011.1 ) software . the complete 850k and 450k data from all the described samples are available for download from ncbi geo . cpg markers present on methylationepic were classified based on its chromosome location , the infinium chemistry used to interrogate the marker ( infinium i , infinium ii ) and the feature category gene region as per ucsc annotation ( tss200 , tss1500 , 5utr , 1st exon , body , 3utr ) . related to this last classification , categories included tss200 as the region between 0 and 200 bases upstream from the transcriptional start site ( tss ) ; tss1500 category , 2001500 bases upstream tss ; 5utr included the region between the tss and the start site ( atg ) ; cpgs within the first exon of a gene were considered as 1st exon category ; cpgs downstream the first exon including intronic regions until the stop codon , were classified as gene body ; cpgs located downstream the stop codon until the poly a signal were considered as 3utr ; and cpgs that were not classified in any of the previous categories were annotated as intergenic . when multiple genes or tss were associated with a cpg site , category prioritization was applied following a 5-prime to 3-prime criteria ( tss200 > tss1500 > 5utr > 1st exon > body > additional criteria included the location of the marker relative to the cpg island ( open sea , island , shore , shelf ) , fantom 5-associated enhancer regions and regulatory regions described on encode project   such as transcription binding site sequences , open chromatin regions and digital dnase i hypersensitivity clusters . our results validate the infinium methylationepic beadchip ( 850k ) as a highly reliable genomic platform for the study of dna methylation patterns and levels in the human genome . most importantly , in addition to showing great correlation with previous dna methylation platforms , high reproducibility and suitability to study formalin - fixed paraffin - embeded samples and 5-hydroxymethylcytosine , it also represents an extremely valuable tool to decipher how dna methylation changes in unexplored territories , such as enhancer sequences , contribute to cell homeostasis and human diseases . the obtention of full human dna methylomes requires a large effort in time and budget . user - friendly dna methylation microarrays , such as the illumina infinium humanmethylation450 beadchip ( 450k ; illumina inc . , ca , usa ) , have become widely adopted , but many potentially important regulatory cpg sites are not interrogated . this study investigates whether the newly developed methylationepic beadchip ( 850k ) can be a reliable tool for more comprehensive dna methylation analyses . the recently developed epic microarray interrogates the methylation status of 853,307 cpg sites , adding 413,745 cpg positions in comparison to the 450k . the new cpgs in the 850k microarray are enriched in regulatory regions provided by the encode and fantom5 projects such as enhancers , transcription factor binding sites ,  open chromatin regions and dnase i hypersensitive regions . dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected using the 450k microarray . replication experiments by hybridizing twice at 850k the same sample detected completely interchangeable cpg methylation values . methylation levels detected from the same dna sample obtained from fresh tissue or formalin - fixing paraffin - embedding were highly correlated . the samples included in the study were all from human origin including fresh - frozen and formalin - fixed paraffin - embedding . dna extraction , bisulfite conversion , site - specific oxidation and array hybrydization to the 450k and 850k microarrays was performed according to the manufacturer instructions ( illumina inc . ) . the recently developed epic microarray interrogates the methylation status of 853,307 cpg sites , adding 413,745 cpg positions in comparison to the 450k . the new cpgs in the 850k microarray are enriched in regulatory regions provided by the encode and fantom5 projects such as enhancers , transcription factor binding sites ,  open chromatin regions and dnase i hypersensitive regions . dna methylation data obtained with the new 850k microarray was highly correlated with the methylation levels detected using the 450k microarray . replication experiments by hybridizing twice at 850k the same sample detected completely interchangeable cpg methylation values . methylation levels detected from the same dna sample obtained from fresh tissue or formalin - fixing paraffin - embedding were highly correlated . the samples included in the study were all from human origin including fresh - frozen and formalin - fixed paraffin - embedding . dna extraction , bisulfite conversion , site - specific oxidation and array hybrydization to the 450k and 850k microarrays was performed according to the manufacturer instructions ( illumina inc . ) .