BACKGROUND
sequence capture enrichment strategies and single molecule sequencing  are expected to increase the rate of gene discovery for genetically heterogeneous diseases.
there have been several recent reports on the successful application of sms to interrogate both viral  <cit>  and bacterial  <cit>  genomes.
at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing in humans.
recently, the targeted exon sequencing of  <dig> cancer gene mutations from tumor/blood samples using the pacbio rs platform was reported indicating that achieving longer reads with sms was feasible in human samples  <cit> .
in that study, target enrichment was achieved through the generation of amplicons averaging 340 bp in length.
more than one million couples worldwide seek reproductive assistance each year because of infertility  <cit> .
unfortunately, infertility therapy involving controlled ovarian stimulation  may result in potentially fatal iatrogenic ovarian hyperstimulation syndrome .
ohss reported as leading cause of maternal mortality in uk  <cit> .
the overarching objective of this study was to identify predictive genetic biomarkers for outcome to controlled ovarian hyperstimulation .
patient response to coh is variable and likely influenced by a diverse array of genetic  factors requiring sophisticated next-generation sequencing  techniques for elucidation.
to date, there have been no tools developed to query all regions  of candidate genes for coh and its major iatrogenic complication, ovarian hyperstimulation syndrome .
we have developed a targeted sms  panel containing  <dig> loci that have been implicated in either response to coh or ohss.
our approach utilized droplet-based emulsion pcr for the generation of  <dig> amplicons averaging 1 kb in length for t-sms.
we report the successful development and implementation of this novel technique and an offer proof of concept of its utility.
methods
sample collection and processing
this study was approved by george washington university institutional review board.
it was open to all adult  female patients recruited previously treated or currently seeking ohss treatment at the gw fertility and ivf center.
written informed consent for participation was obtained from the participants prior to sample collection.
wet lab and dry lab work carried out within genetics in medicine research institute of children’s national medical center.
ovarian hyperstimulation syndrome, non-responders and hyper-responders to ovarian simulation were defined clinically based on the criteria established by navot  <cit> .
approximately 5 ml of blood was collected for genetic analysis.
total genomic dna was extracted from  <dig> ul edta anti-coagulated venous blood using magnetic beads  on a fully automated system.
dna  quality and quantity was measured  using a qubit dsdna hs assay kit and system .
samples showing rna contamination  were discarded and samples with a 260/ <dig> ratio less than one were excluded from further processing.
dna was quantified in duplicate for each sample and the mean value was used for further calculations.
dna integrity  was analyzed using an e-gel® agarose gel electrophoresis system on a  <dig> % agarose gel.
samples showing fragmentation were discarded and re-isolated from fresh patient samples.
dna  was sonicated using a covaris s <dig> system  for 180 s to ~5 kb fragment size .
target gene list and primer design
the target gene list was developed from an extensive literature search.
it included genes that were a) implicated in coh response, b) associated with ohss or c) regulated gonadotropin action and/or ovarian angiogenesis.
genes included in the target list either a) harbored variants associated with coh outcome, b) displayed differential expression  in ohss and/or 3) played a significant role in gonadotropin signaling or in regulating vascular permeability in the ovary.
the genomic targets were comprehensive and included intronic, exonic, 5′ and 3′ untranslated regions  of the target genes.
the total targeted sequence consisted of  <dig>  mb covering  <dig> loci with 1x tiling .
the primer library was designed using the manufacturer’s parameters  and all primers were first tested with primer <dig> .
the primer design pipeline performed an exhaustive primer selection across all of the regions submitted and generated  <dig> unique amplicons  using  <dig> primers .
repeat masking was not performed on the input regions to the primer design pipeline.
primers were designed to provide ~100 bp overlap between adjacent amplicons and avoided primer binding to snps and repeat regions.
there was no allele dropouts discovered in the final design.
droplet-based multiplex amplification
amplification was carried out similar to previously described methods  <cit>  and according to the manufacturer’s protocol.
following amplification each pcr emulsion was broken to release individual amplicons from the pcr droplets and samples were purified using a minelute column  following the manufacturer’s recommended protocol.
purified amplicons were then tested on an agilent bioanalyzer  and qubit  to assure quality and quantity and confirm that the amplicon profile matches the expected histogram profile.
library construction and single molecule, real-time sequencing
amplicons  were converted to smrtbell™ templates using the pacbio® rs dna template preparation kit , incubated for 15 min at 25°c and further purified with a  <dig> x ampure xp clean-up kit and eluted in 30 μl buffer.
blunt adapters were ligated to each amplicon to facilitate circle replication  and to permit error control by calculating the consensus .
exonuclease incubation was carried out in order to remove all unligated adapters.
samples were extracted twice  and the final “smrt bells” were eluted in 10 μl eb.
final quantification was carried out on an agilent  <dig> bioanalyzer with 1 μl of library.
the amount of primer and polymerase required for the binding reaction was determined using the smrt bell concentration  and insert size previously determined using the manufacturer-provided calculator.
primers were annealed and polymerase was bound using the dna/polymerase binding kit  <dig>  .
the complexes were stored at −20°c or diluted for immediate sequencing.
sequencing mixes were diluted to the required concentration with the manufacturer provided dilution buffer prior to loading onto 96-well plates.
sample plates were loaded onto the instrument along with the dna sequencing kit  <dig>  .
sequencing was performed using pacbio smrt sequencing technology as previously described  using a smrt cell 8pac.
in all sequencing runs,  <dig> x 45 min movies were captured for each smrt cell loaded with a single binding complex.
data analyses
the data analyses pipeline is provided in table  <dig>  primary filtering analysis was first performed using the pacbio rs server prior to data being transferred to the smrt portal using the smrt analysis pipeline version  <dig> .
<dig> ; http://www.pacificbiosciences.com/products/pacificbio-rs-workflow-main/).
secondary analysis was conducted using the genome analysis toolkit   embedded in the smrt portal.
output files  were transferred to wannovar  for variant  calling .
the project was registered with the nih bioproject database .
all sequence data was made accessible from the nih next generation sequence read archive  data base .table  <dig>
data pipeline
primary analyses
q metrics
smrt standard pipeline
secondary analyses
gatk
annovar
data visualization
sanger validation of variants
validation of sms variants was conducted by sanger dna sequencing as previously described  <cit> .
primers were designed using the national center for biotechnology information website  and university of santa cruz genome browser .
multiple sequence alignments were carried out using chromaspro software .
all variants were reported according to standard nomenclature.
RESULTS
single molecule sequencing of dna libraries
we targeted the entire coding region  and the 3′ and 5′ utr non-coding sequences of  <dig> candidate loci covering ~ <dig>  mb per sample.
our primer design yielded  <dig> primer pairs that generated  <dig> amplicons that were confirmed to be 1 kb in length .
amplicons were tiled to have an average overlap of  <dig> base pairs  to facilitate coverage and assembly.
for the sms-generated raw reads the average read length was  <dig> nucleotides  and ~5% were >6000 nt.
sms  was successful in capturing 100% sequence information from  <dig> out of the  <dig> amplicons targeted .
after filtering for circular consensus  reads, the mean read length was 3200 nt which was likely due to the use of a longer sequencing protocol to accommodate the larger size  of the amplicons .
the mean mapped ccs read accuracy was 97%.
a small percentage  of consensus reads of were >6215 nt.table  <dig>
characteristics of captured sequence
characteristic
result
we generated average 900 bp mean mapped subreads with a mean zero-mode waveguide  occupancy of 85%.
in our primary design, we calculated target coverage depth using the manufacturer’s formula .
based on this, we used  <dig> chips per sample and sms data were collected in  <dig> x 45-min movies to attain 17x targeted ccs coverage depth.
these results are in agreement with recent targeted sequencing studies shown to have higher coverage depth than exome and whole genome sequencing using different enrichment strategies  <cit> .
previous work has suggested that 10x ccs coverage depth  can accurately  distinguish between heterozygous and homozygous snps  <cit> .
consequently, our filtering was set to a minimum cut-off of  <dig> x ccs coverage depth.
repetitive sequences
guanine-cytosine -rich regions of the genome pose significant challenges for high-throughput dna sequencing.
we were able to sequence 1 kb amplicons generated from gc-rich regions in our targeted sequences.
moreover, we were able to align these gc-rich sequences with similar success as non-gc-rich regions which is consistent with other reports  <cit> .
as proof of concept, figure  <dig> shows tiled coverage of the vegfa gene using total  <dig> amplicons .
vegfa has ~72% gc content .
the uniform coverage for three representative samples aligned against the hg <dig> reference sequence is provided in the circos plot in figure  <dig> figure  <dig>
read length vs. gc coverage.
this is a representative example of typical read length and coverage  of gc rich regions  and sequence results imported as custom track in ucsc genome browser.
fragments  were tiled to cover the entire genomic region of the vegfa gene .
there were a total of  <dig> amplicons  with 50–150 bp overlap.
b. screen capture showing the high gc content  of the vegfa gene which was successfully 005b1:1] sequenced.
uniform coverage between amplicons using droplet pcr with sms technology.
circos plot illustrating the relative coverage of target sequences in  <dig> samples.
outermost blue displays target genomic sequence with respect to chromosomal location.
for each target sequence, the percentage gc content is provided in purple .
the bases covered for bait regions are shown in green .
the coverage of sms for  <dig> representative samples is provided in red.
confirmation of t-sms base calls
base-call accuracy is of great concern with next generation sequencing technologies.
we have validated  <dig> snps identified by t-sms with sanger dna sequencing.
for those exonic variants identified by t-sms,  <dig> of  <dig>  of the snps were verified by sanger dna sequencing.
as an example, figure  <dig> shows the validation of rs <dig> in the luteinizing hormone choriogonadotropin receptor  gene.
this missense variant  passed all the filters and variant annotation analyses steps  and was further validated using conventional sanger sequencing  and here we report it in two severe ohss patients.figure  <dig>
validation of rs <dig> identified by t-sms.
smrt® view screenshot of secondary data analysis from smrt® portal .
validation of the lhcgr rs <dig> heterozygous variant discovered using t-sms by sanger dna sequencing.
the figure depicts the process of biomarker identification by sms  and validation by conventional sanger sequencing .
CONCLUSIONS
targeted sequencing approaches are advantageous for enriching variant identification, simplifying data analysis and avoiding ethical issues surrounding incidental findings.
we have developed a custom protocol and data processing pipeline for generating 1 kb amplicons by emulsion pcr for t-sms.
our preliminary analysis has initially focused on coding region variants in each of our  <dig> candidate loci for ohss.
although smaller amplicons may theoretically yield more readings than larger fragments, we have found that fixed/same size and longer amplicons work effectively with droplet pcr enrichment combined with sms.
employing t-sms technology has provided improved resolution by yielding longer reads and sequencing many target genes in a relatively short period of time .
moreover, t-sms of large amplicons had low composition bias and an error profile that is orthogonal to other next generation sequencing platforms that have promise for clinical diagnosis.
to the best of our knowledge, this is the first study reporting the successful sequencing of 1 kb amplicons utilizing droplet pcr combined with sms technology from human samples.
these data show excellent promise for follow-up studies with a larger number of ohss cases.
availability of supporting data
supporting data is included as additional files.
project details and sequence data registered and can be found in the publicly available databases: http://www.ncbi.nlm.nih.gov/bioproject/193545http://www.ncbi.nlm.nih.gov/pmc/articles/pmc2808927/.
additional file
additional file 1:
targeted gene list.
abbreviations
smssingle molecule sequencing
cohcontrolled ovarian hyperstimulation
ohssovarian hyperstimulation syndrome
ngsnext generation dna sequencing
pcrpolymerase chain reaction
gdnagenomic dna
ccscircular consensus sequencing
smrt®single molecule, real-time
t-smstargeted sms
zmwzero-mode waveguide
snpsingle nucleotide polymorphism
competing interests
the authors declare that they have no competing interests.
authors’ contributions
fs, designed the study and conducted the experiments, data analyses, and drafted the paper.
pg and df identified patients and collected biomaterial.
tjo participated in study design, conducted experiments and analysis and drafted the paper.
ah participated in study design and analysis and drafted the paper.
all authors participated in drafting the paper.
all authors read and approved the final manuscript.

 sequence capture enrichment strategies and single molecule sequencing are expected to increase the rate of gene discovery for genetically heterogeneous diseases. there have been several recent reports on the successful application of sms to interrogate both viral and bacterial genomes. at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing in humans. recently, the targeted exon sequencing of cancer gene mutations from tumor/blood samples using the pacbio rs platform was reported indicating that achieving longer reads with sms was feasible in human samples. in that study, target enrichment was achieved through the generation of amplicons averaging 340 bp in length. more than one million couples worldwide seek reproductive assistance each year because of infertility. unfortunately, infertility therapy involving controlled ovarian stimulation may result in potentially fatal iatrogenic ovarian hyperstimulation syndrome. ohss reported as leading cause of maternal mortality in uk. the overarching objective of this study was to identify predictive genetic biomarkers for outcome to controlled ovarian hyperstimulation. patient response to coh is variable and likely influenced by a diverse array of genetic factors requiring sophisticated next-generation sequencing techniques for elucidation. to date, there have been no tools developed to query all regions of candidate genes for coh and its major iatrogenic complication, ovarian hyperstimulation syndrome. we have developed a targeted sms panel containing loci that have been implicated in either response to coh or ohss. our approach utilized droplet-based emulsion pcr for the generation of amplicons averaging 1 kb in length for t-sms. we report the successful development and implementation of this novel technique and an offer proof of concept of its utility. methods sample collection and processing this study was approved by george washington university institutional review board. it was open to all adult female patients recruited previously treated or currently seeking ohss treatment at the gw fertility and ivf center. written informed consent for participation was obtained from the participants prior to sample collection. wet lab and dry lab work carried out within genetics in medicine research institute of children’s national medical center. ovarian hyperstimulation syndrome, non-responders and hyper-responders to ovarian simulation were defined clinically based on the criteria established by navot. approximately 5 ml of blood was collected for genetic analysis. total genomic dna was extracted from ul edta anti-coagulated venous blood using magnetic beads on a fully automated system. dna quality and quantity was measured using a qubit dsdna hs assay kit and system. samples showing rna contamination were discarded and samples with a 260/ ratio less than one were excluded from further processing. dna was quantified in duplicate for each sample and the mean value was used for further calculations. dna integrity was analyzed using an e-gel® agarose gel electrophoresis system on a % agarose gel. samples showing fragmentation were discarded and re-isolated from fresh patient samples. dna was sonicated using a covaris s system for 180 s to ~5 kb fragment size. target gene list and primer design the target gene list was developed from an extensive literature search. it included genes that were a) implicated in coh response, b) associated with ohss or c) regulated gonadotropin action and/or ovarian angiogenesis. genes included in the target list either a) harbored variants associated with coh outcome, b) displayed differential expression in ohss and/or 3) played a significant role in gonadotropin signaling or in regulating vascular permeability in the ovary. the genomic targets were comprehensive and included intronic, exonic, 5′ and 3′ untranslated regions of the target genes. the total targeted sequence consisted of  mb covering loci with 1x tiling. the primer library was designed using the manufacturer’s parameters and all primers were first tested with primer. the primer design pipeline performed an exhaustive primer selection across all of the regions submitted and generated unique amplicons using primers. repeat masking was not performed on the input regions to the primer design pipeline. primers were designed to provide ~100 bp overlap between adjacent amplicons and avoided primer binding to snps and repeat regions. there was no allele dropouts discovered in the final design. droplet-based multiplex amplification amplification was carried out similar to previously described methods and according to the manufacturer’s protocol. following amplification each pcr emulsion was broken to release individual amplicons from the pcr droplets and samples were purified using a minelute column following the manufacturer’s recommended protocol. purified amplicons were then tested on an agilent bioanalyzer and qubit to assure quality and quantity and confirm that the amplicon profile matches the expected histogram profile. library construction and single molecule, real-time sequencing amplicons were converted to smrtbell™ templates using the pacbio® rs dna template preparation kit, incubated for 15 min at 25°c and further purified with a x ampure xp clean-up kit and eluted in 30 μl buffer. blunt adapters were ligated to each amplicon to facilitate circle replication and to permit error control by calculating the consensus. exonuclease incubation was carried out in order to remove all unligated adapters. samples were extracted twice and the final “smrt bells” were eluted in 10 μl eb. final quantification was carried out on an agilent bioanalyzer with 1 μl of library. the amount of primer and polymerase required for the binding reaction was determined using the smrt bell concentration and insert size previously determined using the manufacturer-provided calculator. primers were annealed and polymerase was bound using the dna/polymerase binding kit. the complexes were stored at −20°c or diluted for immediate sequencing. sequencing mixes were diluted to the required concentration with the manufacturer provided dilution buffer prior to loading onto 96-well plates. sample plates were loaded onto the instrument along with the dna sequencing kit. sequencing was performed using pacbio smrt sequencing technology as previously described using a smrt cell 8pac. in all sequencing runs, x 45 min movies were captured for each smrt cell loaded with a single binding complex. data analyses the data analyses pipeline is provided in table  primary filtering analysis was first performed using the pacbio rs server prior to data being transferred to the smrt portal using the smrt analysis pipeline version.; http://www.pacificbiosciences.com/products/pacificbio-rs-workflow-main/). secondary analysis was conducted using the genome analysis toolkit embedded in the smrt portal. output files were transferred to wannovar for variant calling. the project was registered with the nih bioproject database. all sequence data was made accessible from the nih next generation sequence read archive data base.table data pipeline primary analyses q metrics smrt standard pipeline secondary analyses gatk annovar data visualization sanger validation of variants validation of sms variants was conducted by sanger dna sequencing as previously described. primers were designed using the national center for biotechnology information website and university of santa cruz genome browser. multiple sequence alignments were carried out using chromaspro software. all variants were reported according to standard nomenclature.  single molecule sequencing of dna libraries we targeted the entire coding region and the 3′ and 5′ utr non-coding sequences of candidate loci covering ~  mb per sample. our primer design yielded primer pairs that generated amplicons that were confirmed to be 1 kb in length. amplicons were tiled to have an average overlap of base pairs to facilitate coverage and assembly. for the sms-generated raw reads the average read length was nucleotides and ~5% were >6000 nt. sms was successful in capturing 100% sequence information from out of the amplicons targeted. after filtering for circular consensus reads, the mean read length was 3200 nt which was likely due to the use of a longer sequencing protocol to accommodate the larger size of the amplicons. the mean mapped ccs read accuracy was 97%. a small percentage of consensus reads of were >6215 nt.table characteristics of captured sequence characteristic result we generated average 900 bp mean mapped subreads with a mean zero-mode waveguide occupancy of 85%. in our primary design, we calculated target coverage depth using the manufacturer’s formula. based on this, we used chips per sample and sms data were collected in x 45-min movies to attain 17x targeted ccs coverage depth. these results are in agreement with recent targeted sequencing studies shown to have higher coverage depth than exome and whole genome sequencing using different enrichment strategies. previous work has suggested that 10x ccs coverage depth can accurately distinguish between heterozygous and homozygous snps. consequently, our filtering was set to a minimum cut-off of x ccs coverage depth. repetitive sequences guanine-cytosine -rich regions of the genome pose significant challenges for high-throughput dna sequencing. we were able to sequence 1 kb amplicons generated from gc-rich regions in our targeted sequences. moreover, we were able to align these gc-rich sequences with similar success as non-gc-rich regions which is consistent with other reports. as proof of concept, figure  shows tiled coverage of the vegfa gene using total amplicons. vegfa has ~72% gc content. the uniform coverage for three representative samples aligned against the hg reference sequence is provided in the circos plot in figure  figure read length vs. gc coverage. this is a representative example of typical read length and coverage of gc rich regions and sequence results imported as custom track in ucsc genome browser. fragments were tiled to cover the entire genomic region of the vegfa gene. there were a total of amplicons with 50–150 bp overlap. b. screen capture showing the high gc content of the vegfa gene which was successfully 005b1:1] sequenced. uniform coverage between amplicons using droplet pcr with sms technology. circos plot illustrating the relative coverage of target sequences in samples. outermost blue displays target genomic sequence with respect to chromosomal location. for each target sequence, the percentage gc content is provided in purple. the bases covered for bait regions are shown in green. the coverage of sms for representative samples is provided in red. confirmation of t-sms base calls base-call accuracy is of great concern with next generation sequencing technologies. we have validated snps identified by t-sms with sanger dna sequencing. for those exonic variants identified by t-sms, of of the snps were verified by sanger dna sequencing. as an example, figure  shows the validation of rs in the luteinizing hormone choriogonadotropin receptor gene. this missense variant passed all the filters and variant annotation analyses steps and was further validated using conventional sanger sequencing and here we report it in two severe ohss patients.figure validation of rs identified by t-sms. smrt® view screenshot of secondary data analysis from smrt® portal. validation of the lhcgr rs heterozygous variant discovered using t-sms by sanger dna sequencing. the figure depicts the process of biomarker identification by sms and validation by conventional sanger sequencing.  targeted sequencing approaches are advantageous for enriching variant identification, simplifying data analysis and avoiding ethical issues surrounding incidental findings. we have developed a custom protocol and data processing pipeline for generating 1 kb amplicons by emulsion pcr for t-sms. our preliminary analysis has initially focused on coding region variants in each of our candidate loci for ohss. although smaller amplicons may theoretically yield more readings than larger fragments, we have found that fixed/same size and longer amplicons work effectively with droplet pcr enrichment combined with sms. employing t-sms technology has provided improved resolution by yielding longer reads and sequencing many target genes in a relatively short period of time. moreover, t-sms of large amplicons had low composition bias and an error profile that is orthogonal to other next generation sequencing platforms that have promise for clinical diagnosis. to the best of our knowledge, this is the first study reporting the successful sequencing of 1 kb amplicons utilizing droplet pcr combined with sms technology from human samples. these data show excellent promise for follow-up studies with a larger number of ohss cases. availability of supporting data supporting data is included as additional files. project details and sequence data registered and can be found in the publicly available databases: http://www.ncbi.nlm.nih.gov/bioproject/193545http://www.ncbi.nlm.nih.gov/pmc/articles/pmc2808927/. additional file additional file 1: targeted gene list. abbreviations smssingle molecule sequencing cohcontrolled ovarian hyperstimulation ohssovarian hyperstimulation syndrome ngsnext generation dna sequencing pcrpolymerase chain reaction gdnagenomic dna ccscircular consensus sequencing smrt®single molecule, real-time t-smstargeted sms zmwzero-mode waveguide snpsingle nucleotide polymorphism competing interests the authors declare that they have no competing interests. authors’ contributions fs, designed the study and conducted the experiments, data analyses, and drafted the paper. pg and df identified patients and collected biomaterial. tjo participated in study design, conducted experiments and analysis and drafted the paper. ah participated in study design and analysis and drafted the paper. all authors participated in drafting the paper. all authors read and approved the final manuscript. 
after filtering with circular consensus reads, the mean read length was nucleotides. coverage within gc rich regions was achieved by capturing long genomic dna fragments and reading into regions that flank the capture regions. secondary analysis was conducted using the genome analysis toolkit for snp discovery l and wannovar for functional analysis of variants. target enrichment was carried out using droplet-base multiplex polymerase chain reaction technology designed to yield amplicons averaging 1 kb fragment size from candidate loci. targeted capture enrichment of longer fragments using single molecule sequencing is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. filtered functional variants of were further confirmed using conventional sanger sequencing. we developed a targeted single molecule sequencing panel for genes implicated in ovarian response to controlled ovarian hyperstimulation for infertility. single-molecule sequencingdroplet-based pcremulsion pcrnext generation dna sequencingovarian hyperstimulation syndromeohssissue-copyright-statement© the author 2015 combining emulsion pcr-generated 1 kb amplicons and smrt dna sequencing permitted greater depth of coverage for t-sms and facilitated easier sequence assembly. to the best of our knowledge, this is the first report combining emulsion pcr and t-sms for long reads using human dna samples, and ngs panel designed for biomarker discovery in ohss.
 one of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. targeted capture enrichment of longer fragments using single molecule sequencing is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. we developed a targeted single molecule sequencing panel for genes implicated in ovarian response to controlled ovarian hyperstimulation for infertility.  target enrichment was carried out using droplet-base multiplex polymerase chain reaction technology designed to yield amplicons averaging 1 kb fragment size from candidate loci. the total targeted sequence was  mb per sample. sms was carried out using single molecule, real-time dna sequencing, average raw read length =  nucleotides, 5% of the amplicons > nucleotides). after filtering with circular consensus reads, the mean read length was nucleotides. primary data analyses, alignment and filtering utilized the pacific biosciences® smrt portal. secondary analysis was conducted using the genome analysis toolkit for snp discovery l and wannovar for functional analysis of variants. filtered functional variants of were further confirmed using conventional sanger sequencing. ccs reads were able to accurately detect zygosity. coverage within gc rich regions was achieved by capturing long genomic dna fragments and reading into regions that flank the capture regions. as proof of concept, a non-synonymous lhcgr variant captured in two severe ohss cases, and verified by conventional sequencing.  combining emulsion pcr-generated 1 kb amplicons and smrt dna sequencing permitted greater depth of coverage for t-sms and facilitated easier sequence assembly. to the best of our knowledge, this is the first report combining emulsion pcr and t-sms for long reads using human dna samples, and ngs panel designed for biomarker discovery in ohss. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords single-molecule sequencingdroplet-based pcremulsion pcrnext generation dna sequencingovarian hyperstimulation syndromeohssissue-copyright-statement© the author 2015 