essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) .
it involves placing a small number of large ~710 kbp dna fragments into separate pools .
each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig .
the preparation of each phasing library starts by shearing dna into fragments of approximately 10 kbp in size ; these fragments are gel - purified and ligated with amplification adaptors at both ends .
fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig .
the number of fragments amplified ( 3,0006,000 ) is chosen to minimize the probability of two fragments overlapping , which later simplifies fragment calling in the informatics pipeline .
each resulting pool of amplified molecules is prepared into a sequencing library using the nextera dna transposase , and sequencing adapters with barcodes unique to each well are incorporated through limited - cycle pcr .
the resulting sub - libraries are pooled and sequenced on the illumina platform ( fig .
a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads .
the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each
variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) .
fragments called at this stage have n50 lengths of about 79 kbp ( i.e. at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) .
the relatively short length of these reads compared to older technologies required the development of a haplotyping algorithm , prism ( fig .
1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief ,
first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs .
then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs .
such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . between each local block
, prism produces confidence scores that indicate the likelihood of introducing a switch error owing to statistical phasing . in applications where a high level of accuracy is desired
, the user may introduce breaks in statistically assembled haplotype contigs whenever a confidence score falls below a certain threshold .
the ability to trade - off between accuracy and completeness is a feature of prism that to our knowledge is not provided by other phasing algorithms that we expect to be useful in applications that demand a high a level of precision .
the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary
the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% .
the key algorithmic tool used by prism is a hidden markov model ( hmm ; supplementary material ) . at a high level
, the hmm tries to represent the partially phased haplotypes of the sample as an  imperfect mosaic  of haplotypes from a pre - phased reference panel from the 1000 genomes project .
confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model .
the prism hmm extends the work of li and stephens , on which modern statistical phasing packages such as impute2 ( ref . ) or shape - it are based . unlike existing packages ,
our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods .
the only statistical package able to leverage partial phase information is the recently introduced shapeit2 ; its underlying algorithm follows the same basic approach as prism . as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) .
we evaluated the performance of slrh at different accuracy thresholds by introducing breaks in the global haplotype contigs whenever the confidence score of a local block was below a threshold . depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . 2 and supplementary tables 5 , 6 ) .
in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) .
about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) .
the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants .
the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig .
2 ) . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package .
about 1,500 genes were affected by these mutations , and about 500 were found to have at least one damaging snv on both the maternal and the paternal copy ( table 2 ) . to assess the accuracy of slrh , we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples .
even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . breaking these haplotypes at low - confidence positions
further improved their precision : in every sample , long switch errors occurred at an average rate between 0.2 and 0.9 per mbp , depending on the chosen accuracy threshold ( fig . 2 and supplementary tables 5 , 6 ) . at the 0.9 threshold , long switch events occurred at a rate of 0.47 per mbp for sample na12878 ( supplementary tables 7 , 8) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping .
this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations . finally , the absolute accuracy of the haplotype blocks ( i.e. without correcting for haplotype switching ) varied between 93% and 96% , depending on the sample .
next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library .
we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach .
overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig .
9899% of all snvs were phased in blocks with n50 lengths of 400500 kbp , depending on the sample ( supplementary fig . 3 and supplementary tables 10 , 11 ) .
long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp .
this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample .
finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . 3 and supplementary table 12 ) .
the two libraries were prepared with exactly the same input parameters ( e.g. fragment length , number of fragments per well ) ; their performance metrics differed by less than 1% and the two replicates assigned the same phase to most snvs ( online methods ) .
next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information .
we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) .
we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) .
ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig .
the full list of dmrs and their associated genes is available in the supplementary material . to gather more insight into
how differential methylation may affect gene expression , we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 .
consistently with previous findings , the dmrs were significantly enriched at gene promoters ( p < 2.2e-16 , binomial test ) .
about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig .
we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode .
we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . 4 and 5 ) .
overall , the above findings support the fact that differential methylation plays a role in gene regulation , particularly in the differential expression of genes .
we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) .
we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . since methylc - seq can detect dna methylation in the whole genome
while rrbs only detect dna methylation in gc- enriched regions , our results suggest the prevalence of asm events outside of cpg islands captured by rrbs technology . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles .
we suspect the rrbs technology may introduce high bias from the amplification that leads to high false positive rates .
both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) .
analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate .
a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments .
the long - range switch accuracy on both datasets was similar , but the short switch accuracy was much higher on the synthetic dataset .
this suggests that pcr and nextera mainly introduce gaps in the phased haplotypes as well as point errors at individual variants ; however , their impact on long - rage phase information appears to be small .
essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) .
it involves placing a small number of large ~710 kbp dna fragments into separate pools .
each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig .
the preparation of each phasing library starts by shearing dna into fragments of approximately 10 kbp in size ; these fragments are gel - purified and ligated with amplification adaptors at both ends .
fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig .
the number of fragments amplified ( 3,0006,000 ) is chosen to minimize the probability of two fragments overlapping , which later simplifies fragment calling in the informatics pipeline .
each resulting pool of amplified molecules is prepared into a sequencing library using the nextera dna transposase , and sequencing adapters with barcodes unique to each well are incorporated through limited - cycle pcr .
the resulting sub - libraries are pooled and sequenced on the illumina platform ( fig .
a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads .
the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each
variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) .
fragments called at this stage have n50 lengths of about 79 kbp ( i.e. at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) .
the relatively short length of these reads compared to older technologies required the development of a haplotyping algorithm , prism ( fig .
1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief ,
first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs .
then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs .
such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . between each local block
, prism produces confidence scores that indicate the likelihood of introducing a switch error owing to statistical phasing . in applications where a high level of accuracy is desired
, the user may introduce breaks in statistically assembled haplotype contigs whenever a confidence score falls below a certain threshold .
the ability to trade - off between accuracy and completeness is a feature of prism that to our knowledge is not provided by other phasing algorithms that we expect to be useful in applications that demand a high a level of precision .
the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary
the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% .
the key algorithmic tool used by prism is a hidden markov model ( hmm ; supplementary material ) . at a high level
, the hmm tries to represent the partially phased haplotypes of the sample as an  imperfect mosaic  of haplotypes from a pre - phased reference panel from the 1000 genomes project .
confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model .
the prism hmm extends the work of li and stephens , on which modern statistical phasing packages such as impute2 ( ref . ) or shape - it are based . unlike existing packages ,
our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods .
the only statistical package able to leverage partial phase information is the recently introduced shapeit2 ; its underlying algorithm follows the same basic approach as prism .
as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) .
we evaluated the performance of slrh at different accuracy thresholds by introducing breaks in the global haplotype contigs whenever the confidence score of a local block was below a threshold .
depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . 2 and supplementary tables 5 , 6 ) .
in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) .
about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) .
the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants .
the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig .
2 ) . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package .
about 1,500 genes were affected by these mutations , and about 500 were found to have at least one damaging snv on both the maternal and the paternal copy ( table 2 ) . to assess the accuracy of slrh
, we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples .
even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . breaking these haplotypes at low - confidence positions further improved their precision : in every sample , long switch errors occurred at an average rate between 0.2 and 0.9 per mbp , depending on the chosen accuracy threshold ( fig . 2 and supplementary tables 5 , 6 ) .
at the 0.9 threshold , long switch events occurred at a rate of 0.47 per mbp for sample na12878 ( supplementary tables 7 , 8) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping .
this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations .
finally , the absolute accuracy of the haplotype blocks ( i.e. without correcting for haplotype switching ) varied between 93% and 96% , depending on the sample .
next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library .
we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach .
overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig .
9899% of all snvs were phased in blocks with n50 lengths of 400500 kbp , depending on the sample ( supplementary fig . 3 and supplementary tables 10 , 11 ) .
long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp .
this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample .
finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . 3 and supplementary table 12 ) .
the two libraries were prepared with exactly the same input parameters ( e.g. fragment length , number of fragments per well ) ; their performance metrics differed by less than 1% and the two replicates assigned the same phase to most snvs ( online methods ) .
next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information .
we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) .
we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) .
ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig .
the full list of dmrs and their associated genes is available in the supplementary material . to gather more insight into how differential methylation may affect gene expression
, we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 . consistently with previous findings , the dmrs were significantly enriched at gene promoters ( p < 2.2e-16 , binomial test ) . about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig .
we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode .
we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . 4 and 5 ) .
overall , the above findings support the fact that differential methylation plays a role in gene regulation , particularly in the differential expression of genes .
we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) .
we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . since methylc - seq can detect dna methylation in the whole genome
while rrbs only detect dna methylation in gc- enriched regions , our results suggest the prevalence of asm events outside of cpg islands captured by rrbs technology . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles .
we suspect the rrbs technology may introduce high bias from the amplification that leads to high false positive rates .
both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) .
analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate .
a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments .
the long - range switch accuracy on both datasets was similar , but the short switch accuracy was much higher on the synthetic dataset .
this suggests that pcr and nextera mainly introduce gaps in the phased haplotypes as well as point errors at individual variants ; however , their impact on long - rage phase information appears to be small .
the wealth of information one can obtain from a haplotype - resolved genome promises new advances in both biology and medicine .
compared with existing dilution haplotyping methods , slrh produces haplotypes of equal or greater quality using substantially less sequencing effort ( supplementary table 1 ) .
whereas existing methods require from 110 gbp to 496 gbp of sequencing , slrh requires as little as 30 gbp .
moreover , our method phases up to 99% of all snvs , whereas others exhibit phasing rates of at most 97% , and typically less than 95% .
slrh haplotypes also retain long - range phase information , with n50 lengths of 450560 kbp ; alternative methods have n50 lengths from 350 kbp to 600kbp .
notably , slrh achieves this performance without sacrificing accuracy : long - range switching events occur less than once per mbp on average ( 99.9099.92% long switch accuracy ) . for applications demanding an even higher level of precision
, slrh provides confidence scores that may be used to trade - off haplotype completeness for increased accuracy . at the most stringent thresholds , the method yields short and highly accurate regions that may be valuable in clinical applications
the two components of slrh that enable these advances are a low - bias pcr - based amplification step , and the prism statistical phasing algorithm , which compensates for having relatively little input data .
the two components naturally complement each other : although long fragments can not span across long regions of low heterozygosity , such regions typically exhibit high linkage disequilibrium , and are more amenable to statistical phasing
. the limitations of slrh include the need to use a compute cluster for statistical phasing , and a lower phasing accuracy in statistically - assembled regions ( supplementary table 13 ) .
the statistical component of slrh also can not be applied to species other than human due to the lack of a suitable reference panel .
however , we expect that the molecular component can be applied to species with genomes of at least 100 mbp , which are large enough for long fragments to be sufficiently diluted .
finally , compared to a recently introduced proximity ligation - based method ( haploseq ) , our approach produces shorter , but more complete haplotype blocks . while haploseq phases 81% of snvs in a human genome , slrh phases 99% .
the errors of haploseq mostly affect individual positions without altering the global haplotype structure ; slrh produces much fewer errors , but some of them may introduce long - range switching events .
overall , the two methods appear to have complementary strengths and weaknesses . as an example of the scientific studies that are made possible by slrh , we determined the allele - specific methylation patterns across a phased human genome .
we observed many methylation events , and found that the drms are often associated with cis - regulatory regions . in previous studies ,
differential methylation patterns were determined either by purely statistical methods or from mendelian inheritance rules .
such methods may be inaccurate and may not scale to large studies owing to the need to sequence the parents of every subject . here , we were able to reproduce the work of previous studies ? without relying on parental information or large amounts of sequencing . besides differential methylation studies , haplotype information has applications in many areas of genomics , including : ( i ) the analysis of disorders affected by compound heterozygocity , such as blistering skin , cerebral palsy , deafness and others ; ( ii ) population genetics , where population - specific haplotype blocks are currently resolved using lower - accuracy statistical methods ; ( iii ) the detection of structural variations , which has been shown to benefit from phase information ; ( iv ) the matching of hosts and donors in organ transplantation based on the hla region of the genome ; ( v ) the evolution of genomes across species .
the wide range of these fields highlights the importance of phase information in human genetics .
tools that facilitate access to this information such as ones we presented here will lay the foundation for further advances throughout genomics .
 rapid growth of sequencing technologies has greatly contributed to increasing our understanding of human genetics . yet , in spite of this growth , mainstream technologies have been largely unsuccessful in resolving the diploid nature of the human genome . here 
 we describe statistically aided long read haplotyping ( slrh ) , a rapid , accurate method based on a simple experimental protocol that requires potentially as little as 30 gbp of sequencing in addition to a standard ( 50x coverage ) whole - genome analysis for human samples . using this technology , we phase 99% of single - nucleotide variants in three human genomes into long haplotype blocks of 200 kbp to 1 mbp in length . as a demonstration of the potential applications of our method , we determine allele - specific methylation patterns in a human genome and identify hundreds of differentially methylated regions that were previously unknown . 
 such information may offer insight into the mechanisms behind differential gene expression . 
Results
Statistically aided long read haplotyping
Phasing three genomes using SLRH
Whole-genome phasing from 30 Gbp of sequencing
Determining allele-specific DNA methylation
Effects of PCR and Nextera on haplotyping performance
Discussion
Supplementary Material