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Paper text:
embryos derived from parthenogenetic activation of in vitro matured 
 porcine oocytes were produced , transferred into recipient females , and harvested on day 14 
 of gestation as described elsewhere .
the ivv 
 embryos were produced by artificial insemination according to standard industry practices , 
 and were harvested by uterine flushing on day 14 of gestation as well . upon collection , 
 embryos were snap frozen in liquid nitrogen and stored at 80 c until use .
genomic dna was isolated from the ivv and pa embryos using the allprep dna / rna micro kit 
 from qiagen ( valencia , ca , usa ) , and was stored at 20 c until use .
genomic dna ( 500 ng ) 
 from both embryo types was subjected to bisulfite conversion using the ez dna 
 methylation - gold kit from zymo ( irvine , ca , usa ) , according to the manufacturer 's 
 directions .
the epidesigner software from sequenom was used to design primers for 
 bisulfite pcr ( suppl table s1 : on - line only ) .
the hotstartaq plus master mix kit from 
 qiagen was utilized to generate pcr amplicons from bisulfite - converted genomic dna .
pcr 
 reaction mixes included 20.0 l 2 mastermix , 10.0 l water , 4.0 l coraload loading 
 reagent , 2.0 l bisulfite - converted gdna ( at 20 ng/l ) , and 4.0 l forward / reverse primer 
 mix ( final concentration of 1 m each ) .
cycling parameters included a 5 min initial enzyme activation cycle 
 followed by 40 cycles of 95 c/15 sec  55 c/30 sec  72 c/30 sec and then a single 5 min 
 extension termination step of 72 c. resulting pcr products were purified using the 
 qiaquick pcr purification kit from qiagen and pooled by sample type in equimolar 
 concentrations , then sheared to approximately 100150 bp fragments using the bioruptor 
 from diagenode ( liege , belgium ) .
sequencing library preparation proceeded according to 
 standard single - end sequencing protocols provided by illumina , and both final libraries 
 ( ivv and pa ) were loaded onto a single lane of the genome analyzer iix flow cell at a 
 final combined concentration of 7 pm .
sequencing consisted of 240 bp cycles , resulting in 
 a final read length of 80 bp .
sequencing reads were aligned to reference sequences using the bisulfite sequencing 
 algorithm of the novoalign software from novocraft ( selangor , malaysia ) . aligned
reads 
 were uploaded into geneious , a sequence manipulation software product from biomatters 
 ( auckland , new zealand ) , and the snp detection algorithm was used to determine the 
 frequencies of methylated versus unmethylated cytosines in cpg context in the distinct 
 samples for each pcr product . to compare with the results of traditional sequencing ( see 
 below ) ,
the methylation status of the flt1 locus was determined by selecting short reads 
 that aligned perfectly to the target sequence ( n = 54 reads for pa ; n = 52 reads for ivv ) , 
 and evaluating the methylation status of each read using bisma , a web - based dna 
 methylation analysis platform .
traditional bisulfite sequencing to validate the deep sequencing data was performed by 
 subcloning four of the same pcr products used for illumina sequencing ( bcor , 
 cdc42bpb , cdx2 , flt1 ) in to the pgem t - easy 
 vector from promega ( madison , wi , usa ) , transforming subcloned plasmids into e. cloni 10 g 
 chemically - competent bacterial cells ( lucigen ; middleton , wi , usa ) , and sequencing the 
 resulting plasmid preparations .
a minimum of seven unique plasmid preparations were 
 sequenced for each sample / amplicon combination .
the power analysis calculations were performed using software written and made freely 
 available online by dr .
repeated power tests to compare two proportions were performed , 
 changing sample sizes and expected differences between samples with each iteration .
alpha 
 (  ) at 0.05 and equal sample sizes per treatment group were set for each permutation .
the 
 null hypothesis to be tested in each instance was that the two proportions ( % methylated ) 
 were equal ( p1 = p2 ) .
for each set of hypothetical experimental parameters , a power 
 statistic was generated and recorded to create the table of statistical power in fig .
embryos derived from parthenogenetic activation of in vitro matured 
 porcine oocytes were produced , transferred into recipient females , and harvested on day 14 
 of gestation as described elsewhere .
the ivv 
 embryos were produced by artificial insemination according to standard industry practices , 
 and were harvested by uterine flushing on day 14 of gestation as well . upon collection , 
 embryos were snap frozen in liquid nitrogen and stored at 80 c until use .
genomic dna was isolated from the ivv and pa embryos using the allprep dna / rna micro kit 
 from qiagen ( valencia , ca , usa ) , and was stored at 20 c until use .
genomic dna ( 500 ng ) 
 from both embryo types was subjected to bisulfite conversion using the ez dna 
 methylation - gold kit from zymo ( irvine , ca , usa ) , according to the manufacturer 's 
 directions .
the epidesigner software from sequenom was used to design primers for 
 bisulfite pcr ( suppl table s1 : on - line only ) .
the hotstartaq plus master mix kit from 
 qiagen was utilized to generate pcr amplicons from bisulfite - converted genomic dna .
pcr 
 reaction mixes included 20.0 l 2 mastermix , 10.0 l water , 4.0 l coraload loading 
 reagent , 2.0 l bisulfite - converted gdna ( at 20 ng/l ) , and 4.0 l forward / reverse primer 
 mix ( final concentration of 1 m each ) .
cycling parameters included a 5 min initial enzyme activation cycle 
 followed by 40 cycles of 95 c/15 sec  55 c/30 sec  72 c/30 sec and then a single 5 min 
 extension termination step of 72 c. resulting pcr products were purified using the 
 qiaquick pcr purification kit from qiagen and pooled by sample type in equimolar 
 concentrations , then sheared to approximately 100150 bp fragments using the bioruptor 
 from diagenode ( liege , belgium ) .
sequencing library preparation proceeded according to 
 standard single - end sequencing protocols provided by illumina , and both final libraries 
 ( ivv and pa ) were loaded onto a single lane of the genome analyzer iix flow cell at a 
 final combined concentration of 7 pm .
sequencing consisted of 240 bp cycles , resulting in 
 a final read length of 80 bp .
sequencing reads were aligned to reference sequences using the bisulfite sequencing 
 algorithm of the novoalign software from novocraft ( selangor , malaysia ) . aligned
reads 
 were uploaded into geneious , a sequence manipulation software product from biomatters 
 ( auckland , new zealand ) , and the snp detection algorithm was used to determine the 
 frequencies of methylated versus unmethylated cytosines in cpg context in the distinct 
 samples for each pcr product . to compare with the results of traditional sequencing ( see 
 below ) ,
the methylation status of the flt1 locus was determined by selecting short reads 
 that aligned perfectly to the target sequence ( n = 54 reads for pa ; n = 52 reads for ivv ) , 
 and evaluating the methylation status of each read using bisma , a web - based dna 
 methylation analysis platform .
traditional bisulfite sequencing to validate the deep sequencing data was performed by 
 subcloning four of the same pcr products used for illumina sequencing ( bcor , 
 cdc42bpb , cdx2 , flt1 ) in to the pgem t - easy 
 vector from promega ( madison , wi , usa ) , transforming subcloned plasmids into e. cloni 10 g 
 chemically - competent bacterial cells ( lucigen ; middleton , wi , usa ) , and sequencing the 
 resulting plasmid preparations .
a minimum of seven unique plasmid preparations were 
 sequenced for each sample / amplicon combination .
the power analysis calculations were performed using software written and made freely 
 available online by dr .
repeated power tests to compare two proportions were performed , 
 changing sample sizes and expected differences between samples with each iteration .
alpha 
 (  ) at 0.05 and equal sample sizes per treatment group were set for each permutation .
the 
 null hypothesis to be tested in each instance was that the two proportions ( % methylated ) 
 were equal ( p1 = p2 ) . for each set of hypothetical experimental parameters ,
a power 
 statistic was generated and recorded to create the table of statistical power in fig .
abstract   the purpose of this experiment was to implement and evaluate the effectiveness of a 
 next - generation sequencing - based method for dna methylation analysis in porcine embryonic 
 samples . 
 fourteen discrete genomic regions were amplified by pcr using bisulfite - converted 
 genomic dna derived from day 14 in vivo - derived ( ivv ) and parthenogenetic 
 ( pa ) porcine embryos as template dna . 
 resulting pcr products were subjected to 
 high - throughput sequencing using the illumina genome analyzer iix platform . 
 the average 
 depth of sequencing coverage was 14,611 for ivv and 17,068 for pa . 
 quantitative analysis 
 of the methylation profiles of both input samples for each genomic locus showed distinct 
 differences in methylation profiles between ivv and pa samples for six of the target loci , 
 and subtle differences in four loci . 
 it was concluded that high throughput sequencing 
 technologies can be effectively applied to provide a powerful , cost - effective approach to 
 targeted dna methylation analysis of embryonic and other reproductive tissues .