BACKGROUND
since the pioneering publication of margulies et al.
<cit>  many researchers have demonstrated the versatility of the massively parallel  <dig> pyrosequencing technology.
the method has been successfully applied to a large diversity of targets such as nuclear and organellar genomes  <cit> , transcriptomes  <cit> , cloned dna   <cit> , and pcr products   <cit> .
this broad spectrum is complemented by a wide range of analyses, including epigenetic features  <cit> , genome diversity  <cit>  and ancient dna  <cit> .
in principle, the  <dig> technology should also be applicable for haplotyping copy number  variable loci such as the beta-defensin gene cluster  at human chromosome 8p <dig>  .
this locus is intensively studied  <cit>  is recognized as one of the most dynamic regions of the human genome  <cit>  and was proven to be variable in cn as a whole  <cit> .
individual defb cns and specific defb haplotypes have been shown to be associated with psoriasis, crohn's disease and prostate cancer  <cit> .
within the  <dig> defbs assembled in the human reference sequence , a total of  <dig>  single nucleotide polymorphisms  are annotated in dbsnp  which we address, more appropriately, as multisite variations   <cit> .
a potential method to assess individual haplotypes at such loci is the amplification of an msv-containing region by pcr  <cit> .
subsequently, the amplicons are subcloned in plasmids, individual bacterial clones are sanger sequenced and haplotypes are determined according to the bases at the polymorphic positions.
this approach, however, has inherent problems.
first, individual cns can vary between  <dig> and  <dig> defb copies per diploid genome.
theoretically, each genotype can comprise a number of haplotypes equal to this cn.
for individuals with many copies there is a high risk of missing rare haplotypes due to the decreased per-copy sequencing depth of 100- <dig> reads per genotype in a typical cloning/sanger sequencing approach.
second, pcr on complex templates may result in chimera formation by pcr-mediated recombination, creating artificial, false-positive haplotypes  <cit> .
third, the bacterial cloning step may introduce a systematic bias in the observed haplotype frequencies due to differences in the compatibility of clones with the host's lifecycle.
to minimize the risk of spurious haplotyping by pcr/subcloning, independent pcr products have to be analyzed and large numbers of clones should be sequenced.
this necessary quality control is usually too costly and labour intensive for high-throughput applications.
in addition to assessing sequence variations, haplotyping has also been used for the measurement of defb cns  <cit> .
absolute cns, however, cannot be determined and even reliable relative cn measurement by this method is challenging.
e. g. in the simplest case of just  <dig> copies and  <dig> haplotypes in an msv spanning region, relatively small numerical deviations from the theoretical ratio of  <dig>   may result in quite different minimal cn estimations, as  <dig>   indicates  <dig> copies,  <dig>   indicates  <dig> and  <dig>   indicates  <dig>
for effective and accurate determinations of absolute defb cn, multiplex ligation-dependent probe amplification   <cit> , real-time pcr  <cit> , multiplex amplifiable probe hybridisation combined with restriction enzyme digest variant ratios   <cit>  and different variants of paralog ratio tests   <cit>  have been applied, however, they do not provide haplotype information.
knowledge, however, of both defb cn  <cit>  and haplotypes  <cit>  is of great importance with respect to clinically important phenotypes.
therefore, an ideal method for defb analysis and association studies would provide both cns and haplotypes at once.
application of massively parallel  <dig> technology to pcr amplicons allows extreme in-depth sequencing resulting from the huge amount of data generated by a single instrument run.
given a  <dig> bp amplicon and assuming  <dig>  reads of ~ <dig> bp length per 1/16th segment of a gs flx sequencer picotiterplate ,  <dig> pcr products can be sequenced in parallel with  <dig> × coverage.
this over-sequencing can be reasonably reduced using a multiplex approach, pooling either different amplicons generated from the same dna  <cit>  or the same amplicons derived from multiple dnas using a barcoding strategy  <cit> .
however, in the example above, pooling of  <dig> amplicons should still lead to a mean coverage of  <dig> .
this amount of sequences per individual amplicon is still sufficient to reduce the probability for haplotyping errors as described above.
furthermore,  <dig> amplicon sequencing does not require bacterial cloning, avoiding the danger of respective biases.
as result, it delivers a multiple of individual sequences for a given amplicon compared to a routine cloning/sanger sequencing strategy.
we took advantage of this methodological approach for defb haplotyping and cn estimation by  <dig> sequencing to study the defb locus in  <dig> dna samples from publicly available lymphoblastoid cell lines .
the results are compared to those obtained by the cloning/sanger approach, and to cn measurements using mlpa, maph/redvr and prt.
methods
dna
genomic dna from the ceph resources , the dna polymorphism discovery resource collection  and the  <cit>   were purchased at the coriell cell depository http://ccr.coriell.org/.
dnas c <dig>  c <dig> and c <dig> were obtained from the european collection of cell cultures ecacc  <cit> .
all  <dig> dnas are derived from human lcls, which were established from individuals of different ancestry, i.e.
caucasian , yoruba  and han chinese .
for na <dig>  na <dig>  and na <dig> no information about the ethnicity of the donors is provided by the discovery resource collection.
<dig> amplicon sequencing
pcr methods have been established to generate  <dig> defb specific amplicons  using  <dig> adaptors a and b containing fusion primers .
pcrs were carried out with  <dig> ng dna using the "advantage" mixed polymerase containing both taq and a proofreading enzyme  and  <dig> cycles of 94°c for  <dig> s, 57°c for  <dig> min and 72°c for  <dig> min followed by 72°c for  <dig> min.
concentrations of the pcr products were measured by nanodrop and the quant-it® picogreen® ds dna assay .
all amplicons derived from an individual dna sample were mixed in an equimolar ratio .
the pools were diluted and subjected to emulsion pcr following the flx empcr protocol for amplicons  using both empcr kits ii  and iii  and sequenced on a gs flx  by both primers on  <dig> lane/pool of a 16-lane gasket on a  <dig> ×  <dig> flx picotiterplate.
the sequences from  <dig> lane were aligned to a backbone consisting of the merged pcr target regions derived from the human reference sequence  and haplotype calls  were inferred using the gs amplicon variant analyzer software .
for verification, amplicon f <dig>  was generated from na <dig> and na <dig> by a separate pcr using the same fusion primers and conditions as described for the amplicon pools.
gs flx sequencing was done on  <dig> lanes/amplicon of a 16-lane gasket on a  <dig> ×  <dig> picotiterplate.
to further increase sequencing depth, a third gs flx run was performed with pooled amplicons f <dig>  f <dig>  f <dig> and f <dig> of na <dig>  na <dig>  na <dig>  and na <dig> using a 4-lane gasket on a  <dig> ×  <dig> picotiterplate.
to avoid misinterpretations caused by incidental chimera formation early in pcr,  <dig> independent reactions were carried out with each primer pair and dna sample.
amplicon subcloning in bacteria and sanger sequencing
using  <dig> out of the  <dig> dnas ,  <dig> pcr products  were generated using unmodified primers .
pcrs were performed with  <dig> ng dna using taq polymerase .
amplification was achieved using  <dig> cycles of 95°c for  <dig> s, 56°c for  <dig> s and 72°c for  <dig> min followed by  <dig> cycles of 95°c for  <dig> s, 58°c for  <dig> s and 72°c for  <dig> min, and finally 72°c for  <dig> min.
pcr products were subcloned into pcr <dig> -topo vector  and  <dig> individual clones per dna and amplicon were sequenced by dye terminator sequencing chemistry using an abi3730xl automated sequencer  and m13rev universal primers.
haplotypes were inferred by manual inspection of phrap alignments using gap <dig> .
other copy number measurement methods
the pyrosequencing based prt , the mlpa analyses  <cit>  and the 5-prt  <cit>  were carried out as previously described.
results of cns measured by maph/redvr and another prt  <cit>  were kindly provided by j. armour and e. hollox.
RESULTS
haplotyping by  <dig> amplicon sequencing and comparison with the cloning/sanger approach
the amplicons used in the present study were designed in order to cover a broad spectrum of regions within defb encompassing putative promoters, exons and introns and intergenic parts.
if possible, amplicon lengths were restricted to span not much more than  <dig> bp in order to obtain consistent haplotype information by  <dig> reads from both ends.
furthermore, the amplified regions were selected to contain a high number of known msvs.
application of these criteria resulted in  <dig> amplicons  with lengths of 194- <dig> bp, spread over a region of  <dig> kb  and harbouring  <dig> known msvs .
each amplicon harbours between  <dig>  and  <dig> msvs .
eleven genomic dnas were selected to represent a broad range of cns as determined by mlpa, that is, 2- <dig> defb copies per diploid genome.
pcrs were performed separately for each dna and primer pair followed by equimolar pooling of all pcr products of the same dna.
for validation purposes,  <dig> additional amplicons from  <dig> samples were analysed by cloning/sanger sequencing  <cit>  .
eleven lanes of a single gs flx run  resulted in  <dig>  reads with an average length of  <dig> bp, producing a total of  <dig>  mb raw data.
of the total reads,  <dig>  reads  could be assembled to the amplicon reference sequences resulting in an average of  <dig>  reads per dna sample and  <dig>  per amplicon .
among the amplicons, f <dig> and f <dig> were underrepresented by  <dig>  and  <dig>  reads, respectively, compared to  <dig> - <dig>  for the remaining  <dig> amplicons.
additionally, the amount of reads per dna sample greatly differed for f <dig> and f <dig> with less than  <dig> reads in  <dig> cases.
therefore, we have excluded the data of f <dig> and f <dig>  leaving a total of  <dig>  reads from  <dig> amplicons used for further analyses of the  <dig> dna samples.
every read may be regarded as representation of a particular defb allele.
a haplotype can be inferred from each read by determination of the msv allele combination, named "haplotype call" .
as amplicons were designed in accordance with the read length achievable by the gs flx chemistry , the small minority of reads greatly shorter than  <dig> bp produced by the flx sequencing may not span the aspired haplotype.
such shorter hcs, however, are less informative and would complicate the analyses.
therefore, these suboptimal hcs were omitted from further analyses, reducing the final data set from  <dig>  reads to  <dig>  most informative hcs .
out of the  <dig> known msvs within the  <dig> amplicons,  <dig> were found to be polymorphic.
for rs <dig> , exclusively the t allele was observed.
in addition, we confirmed in amplicon f <dig> of c <dig>  na <dig> and na <dig> an msv at position  <dig> downstream of defb <dig>  not annotated in dbsnp but present in the hg <dig> reference sequence as a paralogous sequence variation  <cit> .
moreover, we identified a novel base exchange in the same amplicon of na <dig> located  <dig> nucleotides further downstream .
the msv combinations in  <dig> out of  <dig> amplicons could be unambiguously deduced from the overlapping forward and reverse reads.
however, for f <dig>  the read length of the flx chemistry was too short to directly infer complete haplotypes, as the forward reads cover msvs 6- <dig> and the reverse reads 7-10a, respectively .
nevertheless, using data from non-polymorphic samples, the hc ratios in polymorphic samples and a parsimony assumption,  <dig> forward and  <dig> reverse haplotypes collapse consistently into  <dig> complete f <dig> haplotypes .
#: number of hcs; *: alleles in lower cases refer to the  <dig> additionally identified msvs 9a and 10a; (): nominally below 10% threshold; nd: no data.
in total,  <dig> haplotypes could be identified .
the largest variety was found for amplicon f <dig> with  <dig> haplotypes out of  <dig> theoretically possible combinations of msvs.
in contrast, only  <dig> haplotypes were observed for f <dig>  and f <dig> , respectively.
the most abundant haplotypes are for f2: ga, f4: tac, f5: tccgggc, f6: cctcg, f7: tcg and f8: ctaccg,.
tccgggc and tcg were found in all investigated dnas.
joining the haplotypes with the most hcs for each dna sample individually creates  <dig> inferred "long range haplotypes" of which one is present in of  <dig> samples .
remarkably, this inferred haplotype can be generated by just one recombination of physical haplotypes represented by large insert clones from the rpci- <dig> human male bac library, sequenced in the course of the human genome project  <cit>  .
several additional msv combinations were found which are represented by only few reads compared to the haplotypes described above, indicating either the generation of chimeric, pcr-mediated recombinant molecules from different target copies, misincorporations during the emulsion pcr, sequencing errors or cross-contaminations.
in total,  <dig> out of  <dig>  <dig> pcrs and  <dig> out of  <dig> sanger pcrs contained this phenomenon.
based on the estimation by alternative methods that the maximal defb cn in our sample set is  <dig>  we discarded hcs with fractions < <dig>   as such artefacts.
this led to the exclusion of  <dig>   and  <dig>  hcs from the  <dig> and sanger datasets, respectively .
comparing the haplotyping results for both methods, the same haplotypes were found in 85%  except for amplicons f5/s <dig> in samples na <dig> and na <dig> .
in both cases rare  <dig> haplotypes were not found in the sanger reads.
due to the low overall number of f <dig> sequences for na <dig>  the hc frequency for the haplotype inferred from msvs 6- <dig> is lower < <dig>  and had to be excluded .
in order to finally confirm or reject the questionable haplotypes, we generated additional f <dig> amplicons from the  <dig> dna samples and performed  <dig> sequencing in 2/16th plate per sample resulting in  <dig>  and  <dig>  sequences, respectively.
from these reads more than  <dig>  hcs per sample for msvs 6- <dig> and over  <dig>  calls for msvs 7-10a were obtained for each of the  <dig> dnas, unequivocally confirming the presence of the questionable ctgggac and ccgggac haplotypes, respectively .
#: number of hcs; f = hc frequency; *: alleles in lower cases refer to the  <dig> additionally identified msvs 9a and 10a.
determination of relative defb copy numbers by haplotype calls
in order to evaluate the applicability of  <dig> hcs for the measurement of relative defb cns, we calculated the hc ratios for all amplicons with more than one haplotype and inferred the respective minimal cn.
for example, in f <dig> from c <dig>   <dig> haplotypes  were identified with  <dig>   <dig>  and  <dig> calls, respectively.
this corresponds to a normalized call ratio of 1: <dig> : <dig>  that is essentially 1:3: <dig>  indicating a minimal cn of  <dig>  altogether, by this approach  <dig> cn calculations were done.
for samples with more than one polymorphic amplicon we computed the average of the separate cns or of their least common multiple.
the final minimal cn estimations for the samples range between  <dig> and  <dig> .
armour: prt and maph/redvr  <cit>  and personal communication; 5prt.
pprt  <cit> ; mlpa  <cit> .
~: rounded; n: multiple of the minimal estimated cn; *: cn estimations based on ultra-deep  <dig> sequencing; nd: no data; blank: non polymorphic.
the defb cns of the  <dig> samples haplotyped in this study were previously measured by  <dig> independent experiments using different prts, mlpa and maph/redvr  <cit>  and armour and hollox, personal communication].
for  <dig> of the  <dig> dnas, measurement by the different techniques  resulted in identical cns.
the remaining samples showed disagreements of only one copy, indicating the consistency of the data .
therefore, we took the average of these measurements and compared them with cn estimates from the  <dig> hcs.
for  <dig> samples the  <dig> haplotype-based estimates were in agreement with cns determined by the alternative methods.
for another  <dig> dna samples , these numbers differed only by one copy.
in the case of na <dig>  the estimated minimal cn was half of the absolute one.
for na <dig>   <dig> msv combinations are polymorphic, resulting in cns of  <dig> , 2n  and  <dig> , respectively.
only in this single case, the hc-based minimal cn estimations can not be unambiguously interpreted and considerably deviates from  <dig> copies per diploid genome, consistently determined by  <dig> alternative methods.
as ultra-deep sequencing of na <dig> and na <dig> amplicon f <dig> provided most consistent cn estimations  for samples with high cns, we further validated this approach by performing a third gs flx run with pooled amplicons f <dig>  f <dig>  f <dig> and f <dig> from these dnas.
moreover, we included na <dig>  and na <dig>  .
to avoid false positive hcs resulting from chimera formation early in pcr, we carried out  <dig> independent reactions with each primer pair and dna sample.
accordingly, we lowered the threshold for exclusion of minor haplotype fractions from 10% to 6% to avoid false negative hcs.
altogether, we obtained  <dig>  hcs providing on average ~ <dig>  per amplicon and sample , of which  <dig> % were excluded as potential chimera, sequencing errors or contaminations.
the extraordinary sequencing depth and the lowered filter threshold identified additional haplotypes for na <dig>  and na <dig>  which increased the resolution of the cn estimation .
ultra-deep sequencing-based cn estimations for samples with high cns  tend to be higher than those determined by the established alternative methods.
nd: no data; blank: non polymorphic.
discussion
impaired beta-defensin synthesis has been described in many human diseased states, namely in inflammatory disorders  <cit> .
while association of a disease and defb cn has been demonstrated  <cit> , associations of sequence variants with disease  <cit>  cannot reliably be investigated with established genotyping methods.
hollox  <cit>  observed that it may be the nucleotide state of an msv that actually causes susceptibility to a disease for which association with cn has been found.
if so, cn is only a proxy for causative sequence variants.
however, description of sequence variation in cn variable loci such as defb at 8p <dig> is inherently challenging.
currently, neither the haplotype structure of defbs nor their arrangements on chromosomes  are known.
the present work provides a first glimpse of the defb haplotype complexity through  <dig> amplicon sequencing of selected polymorphic defensin gene fragments.
among the currently available next generation sequencing  approaches, the  <dig> technology is best suited for this purpose as it provides longest reads that in turn produce the longest inferred physical haplotypes.
eleven dna samples derived from lcls were selected to represent a range of 2- <dig> defb copies per diploid genome.
all dnas are publicly available and  <dig> were investigated in the framework of the hapmap project  <cit> .
four of the lcls were established from individuals of caucasian,  <dig> of yoruba and one of chinese ancestry.
about  <dig>  msv hcs were derived from  <dig> amplicons containing 2- <dig> annotated msvs each.
one msv not deposited in dbsnp and a new one were detected in  <dig> samples of yoruba and one of caucasian ancestry, respectively.
theoretically, the  <dig> msvs in  <dig> amplicons altogether could compose  <dig> haplotypes of which we identified  <dig> .
as exemplified by the analysis of the f <dig> amplicon data, hc ratios provided by the  <dig> deep sequencing approach may serve as additional experimental evidence to deduce longer haplotypes than the maximal ngs read length.
with  <dig> msvs in  <dig> amplicons, totalling to  <dig>  bp, the msv density in the investigated regions is approximately  <dig> msv/kb.
this is higher than the overall single nucleotide variation density of the entire defb currently annotated in dbsnp  and reflects the directed selection of the amplified regions to screen a maximum of polymorphic positions within a short distance.
in comparison, for the most sequence variable locus of the human genome, the mhc complex, alignments of  <dig> mhc haplotypes revealed  <dig> snp/kb  with a higher density  in the intragenic regions  <cit> .
for the genomic loci of hla-a, b and c there are  <dig>  polymorphic positions within  <dig>  bp assigned by dbsnp build  <dig>  corresponding to  <dig> snps/kb.
this indicates that in terms of sequence variability, defb is substantially less polymorphic than the hla loci, and that defb together with the analysed amplicons are comparable to the mhc intragenic regions.
the fact that the  <dig> deep-sequencing revealed only one novel msv on the background of  <dig> previously known ones shows that the dbsnp annotation of the analysed regions is quite comprehensive.
on the other hand according to dbsnp, the msv density of defb is about  <dig> times higher than the snp density of the entire human genome .
moreover, currently unknown additional sequence variability may be still hidden in the defb  which may be revealed by future targeted and whole-genome resequencing efforts.
the haplotypes identified by  <dig> sequencing and by cloning/sanger sequencing were identical except for  <dig> cases, which could be resolved by additional  <dig> sequencing.
however, the ratios of haplotypes derived from a given individual amplicon sometimes differ remarkably.
for example, the c <dig> f <dig> haplotypes ccggac and cccggc were revealed by  <dig> sequencing in a ratio of 1: <dig>  but 1: <dig>   by sanger derived reads.
we suspect the explanation is that the number of hcs from sanger sequenced clones is too low for providing reliable results.
moreover, additional bias may be introduced e.g.
by the bacterial cloning step.
theoretically, the more hcs that are available, the more accurately they should reflect the real ratio of the defb copies with particular haplotypes.
this becomes more important as the number of defbs and/or haplotypes per genome increases.
the conclusion is supported by our efforts to resolve the discrepancies between  <dig> and sanger sequencing for na <dig> and na <dig>  the minor haplotypes of the f <dig> amplicon were supported by  <dig> but not by sanger sequencing, probably due to the low amount of sequenced clones.
additional  <dig> hcs finally confirmed the presence of the f <dig> haplotypes under question.
furthermore we noticed an uneven distribution of forward and reverse reads in the  <dig> sequencing of particular amplicons.
this is obvious in fig.
<dig> with a nearly 1: <dig> forward-to-reverse read ratio and is also reflected in the entire data set of amplicon f <dig> with  <dig>  forward but  <dig>  reverse hcs .
such a skew may be caused by unequal sequencing efficiencies due to the direction-specific sequencing primers as well as to differences in base composition and/or motifs between the forward and reverse strands.
thus in the case of non or partially overlapping forward and reverse reads, the resolution of the approach is limited by the less effective sequencing direction  <cit> .
another problem of any pcr-based haplotyping is the occurrence of chimeric products  <cit> .
since both  <dig> amplicon sequencing and the cloning/sanger approach are based on pcr, the formation of such artefacts must be taken into account.
chimeras can be generated during pcr by heteroduplexes of e.g.
a complete copy of one allele with a fragment from another allele.
completion of the partial strand in the elongation step creates chimeric molecules from which chimeric haplotypes may be erroneously determined.
the degree of chimera formation is dependent on various factors such as polymerase, nucleotide/primer concentrations, cycling conditions and amplicon length.
in addition to the formation of chimeric pcr products, nucleotide misincorporations during the emulsion pcr, sequencing errors and cross-contaminations have to be taken into account.
in the current study, we have appointed and discarded an msv combination as artificial if its relative number of call was lower than 10%  or 6% .
this led to the exclusion of less than 3% of all hcs which otherwise would render the analysis impossible.
false negative hcs by this cutoff per definitionem would only affect dna samples with more than  <dig> and  <dig> defb copies, respectively.
cns > <dig> are rarely reported in the literature  <cit> .
nevertheless, these threshold filters may fail if the artificial molecule appears early in pcr.
to avoid respective misinterpretations, independent pcrs were carried out and pooled before sequencing for the third gs flx run.
the estimation of cns from the hc ratios is intrinsically problematic, since ratios provide only minimal cns and any multiple of that can be ruled out only by additional considerations .
moreover, slightly different ratios lead to different cn estimations.
nevertheless, thanks to the high number of ngs hcs, cns could be correctly determined for  <dig> out of  <dig> samples and differed only by one copy per diploid genome for another  <dig> samples .
remarkably, the high hc numbers obtained by ultra-deep gs flx runs  <dig> and  <dig> consistently point towards higher cns than obtained by the widely used prts/mpla .
it is known that the latter methods tend to underestimate the actual cn in case of defb cns > <dig> .
thus, it has to be proven in the future, whether ultra-deep ngs like  <dig> may provide more reliable results than the currently established methods for cases of high cns per diploid genome.
the recently developed mrfast   <cit>  points in the same direction although the sequencing depth of personalized whole-genome sequencing efforts and the  <dig> genomes project is too low to provide sufficient read counts for reliable estimations of high cns in complex regions like defb.
only in one case were the cn estimations inconsistent .
for this, we suppose  <dig> possible reasons.
first, the estimated cn  <dig> results from amplicon f <dig> with a relatively low amount of reads which in case of the forward sequencing direction  caused a random deviation from the 1: <dig> ratio observed for the reverse hcs.
second, 7- <dig> copies were calculated by the ratio of  <dig> haplotypes in amplicon f <dig>  as the  <dig> haplotypes were confirmed by ultra-deep sequencing of pooled independent amplicons, it is unlikely that the minor haplotype  is a chimera.
as the remaining  <dig> amplicons show no sequence variation and are therefore in agreement with the low cn of  <dig> determined by the alternative methods, sample na <dig> may represent in our hands the first example violating the concordance rule of defb  <cit> .
whether the underlying molecular event represents a natural polymorphism or emerged in cell culture remains to be elucidated in the future.
although our results indicate that ngs based haplotyping and cn estimation thereof is a suitable approach to characterize a highly polymorphic and cn variable loci as the defb cluster, this method has some inherent limitations.
first, haplotyping is restricted to those regions in which at least two msvs are located within the read length of the applied ngs technology.
second, equimolar pooling of pcr products is difficult and also the efficiencies of the following emulsion pcr may deviate among the amplicons.
this can result in differing amounts of hcs per amplicon which may prevent reliable haplotyping if too few sequences are obtained .
third, formation of pcr artefacts leading to the identification of "false", i.e.
chimeric haplotypes always has to be considered.
although distinction between "true" and "false" halpotypes is easier with higher sequencing depth it is problematic for samples with high cns.
CONCLUSIONS
deep  <dig> amplicon sequencing is an effective method for parallel haplotyping and cn estimation in highly polymorphic loci such as defb at chromosome 8p <dig>  for the following reasons:
<dig>  there is no bacterial cloning bias as a source of putative errors in haplotype identification.
moreover, in contrast to the cloning/sanger sequencing approach, thousands of hcs per amplicon can easily be performed leading to a higher accuracy in obtained haplotype ratios and cn estimations inferred thereof.
<dig>  calculated on the basis of the consumable's prices for the present study and normalized to the same yield of informative hcs, the costs for  <dig> amplicon sequencing amounts to only ~4% of those for cloning/sanger sequencing.
although this does not even consider the reduced experimental effort it is still too expensive for high-throughput measurements but acceptable for analyses of small cohorts.
<dig>  recently,  <dig> titanium amplicon sequencing protocols and kits became available , increasing achievable read lengths to  <dig> bp with a 2-fold increase of the number of reads per run.
thus, longer amplicons than in our study can be sequenced, encompassing more or more distant msvs.
moreover, as the output of reads also increases, multiplexing different dnas will be feasible by application of barcoding strategies .
<dig>  although the presented  <dig> amplicon sequencing delivers only relative cns and has to be completed by other approaches, future inclusion of reference amplicons from loci with invariable cn will provide absolute defb cns.
deep  <dig> amplicon sequencing can contribute substantially to investigate sequence variants and haplotype structure of cn variable loci.
nevertheless, increases in both ngs read length and output are needed to eventually completely resolve loci like defb at the physical haplotype level.
authors' contributions
st designed the  <dig> experiments and carried out both the  <dig> and sanger experiments, designed by kh.
mg performed the mlpa and prt experiments.
st, ap and ks analyzed the data.
mp, st, jh, pr and ss conceived of the study, and participated in its design and coordination.
st, mp, kh and ks wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
amplicons , primers and their locations in the human genome.
pcr products , primers used for their amplification and chromosomal locations  of the amplified regions.
msvs and amplicons.
analyzed multisite variations , their rs numbers and chromosomal locations according to snpdb build  <dig> and ncbi build  <dig>  , respectively.
numbers of assembled gs-flx reads per amplicon  and dna .
assembled  <dig> sequences from  <dig> gs flx runs per dna and amplicon.
haplotypes and haplotype calls  for gs-flx runs 1- <dig> and pcr/cloning.
haplotypes, haplotype calls  and their fraction per dna and msv combination, derived from gs flx runs  <dig> and  <dig> and sanger sequencing of cloned pcr products.
haplotypes  by msv combinations.
haplotypes and total number and fraction of haplotype summed up from all dnas sequenced in gs flx runs  <dig> and  <dig>  haplotypes by dna.
identified haplotypes per dna and amplicon, highlighting the most abundant haplotypes.
individual defb copy numbers  derived from the ratio of haplotype calls  per msv combination and comparison with cns by other methods.
estimation of of the defb cluster copy number  from the ratio of haplotype calls  per dna and amplicon and comparison of with cns determined by mlpa and different paralogue ratio tests.
haplotypes and haplotype calls  for gs-flx run  <dig>  haplotypes, haplotype calls  and their fraction per dna and msv combination, derived from gs flx run  <dig> .
individual defb copy numbers  derived from the ratio of haplotype calls  per msv combination from run1+ <dig> versus run <dig>  individual defb copy numbers  for  <dig> dnas in comparison between gs flx run 1+ <dig> versus gs flx run  <dig> .
click here for file
acknowledgements
we thank daniela werler, cornelia luge, silke foerste and beate szafranski for excellent technical assistance.
john armour and ed hollox are acknowledged for kindly providing cn data based on maph/redvr and prt.
we thank niels jahn for bioinformatics support as well as roger horton and andrew heidel for critically reading the manuscript.
this work was supported by grants from the bundesministerium für bildung und forschung  and the gp <dig> project of the network of environmental disorders.

 since the pioneering publication of margulies et al. many researchers have demonstrated the versatility of the massively parallel pyrosequencing technology. the method has been successfully applied to a large diversity of targets such as nuclear and organellar genomes, transcriptomes, cloned dna, and pcr products. this broad spectrum is complemented by a wide range of analyses, including epigenetic features, genome diversity and ancient dna. in principle, the technology should also be applicable for haplotyping copy number variable loci such as the beta-defensin gene cluster at human chromosome 8p. this locus is intensively studied is recognized as one of the most dynamic regions of the human genome and was proven to be variable in cn as a whole. individual defb cns and specific defb haplotypes have been shown to be associated with psoriasis, crohn's disease and prostate cancer. within the defbs assembled in the human reference sequence, a total of single nucleotide polymorphisms are annotated in dbsnp which we address, more appropriately, as multisite variations. a potential method to assess individual haplotypes at such loci is the amplification of an msv-containing region by pcr. subsequently, the amplicons are subcloned in plasmids, individual bacterial clones are sanger sequenced and haplotypes are determined according to the bases at the polymorphic positions. this approach, however, has inherent problems. first, individual cns can vary between and defb copies per diploid genome. theoretically, each genotype can comprise a number of haplotypes equal to this cn. for individuals with many copies there is a high risk of missing rare haplotypes due to the decreased per-copy sequencing depth of 100- reads per genotype in a typical cloning/sanger sequencing approach. second, pcr on complex templates may result in chimera formation by pcr-mediated recombination, creating artificial, false-positive haplotypes. third, the bacterial cloning step may introduce a systematic bias in the observed haplotype frequencies due to differences in the compatibility of clones with the host's lifecycle. to minimize the risk of spurious haplotyping by pcr/subcloning, independent pcr products have to be analyzed and large numbers of clones should be sequenced. this necessary quality control is usually too costly and labour intensive for high-throughput applications. in addition to assessing sequence variations, haplotyping has also been used for the measurement of defb cns. absolute cns, however, cannot be determined and even reliable relative cn measurement by this method is challenging. e. g. in the simplest case of just copies and haplotypes in an msv spanning region, relatively small numerical deviations from the theoretical ratio of may result in quite different minimal cn estimations, as indicates copies, indicates and indicates for effective and accurate determinations of absolute defb cn, multiplex ligation-dependent probe amplification, real-time pcr, multiplex amplifiable probe hybridisation combined with restriction enzyme digest variant ratios and different variants of paralog ratio tests have been applied, however, they do not provide haplotype information. knowledge, however, of both defb cn and haplotypes is of great importance with respect to clinically important phenotypes. therefore, an ideal method for defb analysis and association studies would provide both cns and haplotypes at once. application of massively parallel technology to pcr amplicons allows extreme in-depth sequencing resulting from the huge amount of data generated by a single instrument run. given a bp amplicon and assuming reads of ~ bp length per 1/16th segment of a gs flx sequencer picotiterplate, pcr products can be sequenced in parallel with × coverage. this over-sequencing can be reasonably reduced using a multiplex approach, pooling either different amplicons generated from the same dna or the same amplicons derived from multiple dnas using a barcoding strategy. however, in the example above, pooling of amplicons should still lead to a mean coverage of. this amount of sequences per individual amplicon is still sufficient to reduce the probability for haplotyping errors as described above. furthermore, amplicon sequencing does not require bacterial cloning, avoiding the danger of respective biases. as result, it delivers a multiple of individual sequences for a given amplicon compared to a routine cloning/sanger sequencing strategy. we took advantage of this methodological approach for defb haplotyping and cn estimation by sequencing to study the defb locus in dna samples from publicly available lymphoblastoid cell lines. the results are compared to those obtained by the cloning/sanger approach, and to cn measurements using mlpa, maph/redvr and prt. methods dna genomic dna from the ceph resources, the dna polymorphism discovery resource collection and the were purchased at the coriell cell depository http://ccr.coriell.org/. dnas c c and c were obtained from the european collection of cell cultures ecacc. all dnas are derived from human lcls, which were established from individuals of different ancestry, i.e. caucasian, yoruba and han chinese. for na na and na no information about the ethnicity of the donors is provided by the discovery resource collection. amplicon sequencing pcr methods have been established to generate defb specific amplicons using adaptors a and b containing fusion primers. pcrs were carried out with ng dna using the "advantage" mixed polymerase containing both taq and a proofreading enzyme and cycles of 94°c for s, 57°c for min and 72°c for min followed by 72°c for min. concentrations of the pcr products were measured by nanodrop and the quant-it® picogreen® ds dna assay. all amplicons derived from an individual dna sample were mixed in an equimolar ratio. the pools were diluted and subjected to emulsion pcr following the flx empcr protocol for amplicons using both empcr kits ii and iii and sequenced on a gs flx by both primers on lane/pool of a 16-lane gasket on a × flx picotiterplate. the sequences from lane were aligned to a backbone consisting of the merged pcr target regions derived from the human reference sequence and haplotype calls were inferred using the gs amplicon variant analyzer software. for verification, amplicon f was generated from na and na by a separate pcr using the same fusion primers and conditions as described for the amplicon pools. gs flx sequencing was done on lanes/amplicon of a 16-lane gasket on a × picotiterplate. to further increase sequencing depth, a third gs flx run was performed with pooled amplicons f f f and f of na na na and na using a 4-lane gasket on a × picotiterplate. to avoid misinterpretations caused by incidental chimera formation early in pcr, independent reactions were carried out with each primer pair and dna sample. amplicon subcloning in bacteria and sanger sequencing using out of the dnas, pcr products were generated using unmodified primers. pcrs were performed with ng dna using taq polymerase. amplification was achieved using cycles of 95°c for s, 56°c for s and 72°c for min followed by cycles of 95°c for s, 58°c for s and 72°c for min, and finally 72°c for min. pcr products were subcloned into pcr -topo vector and individual clones per dna and amplicon were sequenced by dye terminator sequencing chemistry using an abi3730xl automated sequencer and m13rev universal primers. haplotypes were inferred by manual inspection of phrap alignments using gap. other copy number measurement methods the pyrosequencing based prt, the mlpa analyses and the 5-prt were carried out as previously described. results of cns measured by maph/redvr and another prt were kindly provided by j. armour and e. hollox.  haplotyping by amplicon sequencing and comparison with the cloning/sanger approach the amplicons used in the present study were designed in order to cover a broad spectrum of regions within defb encompassing putative promoters, exons and introns and intergenic parts. if possible, amplicon lengths were restricted to span not much more than bp in order to obtain consistent haplotype information by reads from both ends. furthermore, the amplified regions were selected to contain a high number of known msvs. application of these criteria resulted in amplicons with lengths of 194- bp, spread over a region of kb and harbouring known msvs. each amplicon harbours between and msvs. eleven genomic dnas were selected to represent a broad range of cns as determined by mlpa, that is, 2- defb copies per diploid genome. pcrs were performed separately for each dna and primer pair followed by equimolar pooling of all pcr products of the same dna. for validation purposes, additional amplicons from samples were analysed by cloning/sanger sequencing. eleven lanes of a single gs flx run resulted in reads with an average length of bp, producing a total of mb raw data. of the total reads, reads could be assembled to the amplicon reference sequences resulting in an average of reads per dna sample and per amplicon. among the amplicons, f and f were underrepresented by and reads, respectively, compared to - for the remaining amplicons. additionally, the amount of reads per dna sample greatly differed for f and f with less than reads in cases. therefore, we have excluded the data of f and f leaving a total of reads from amplicons used for further analyses of the dna samples. every read may be regarded as representation of a particular defb allele. a haplotype can be inferred from each read by determination of the msv allele combination, named "haplotype call". as amplicons were designed in accordance with the read length achievable by the gs flx chemistry, the small minority of reads greatly shorter than bp produced by the flx sequencing may not span the aspired haplotype. such shorter hcs, however, are less informative and would complicate the analyses. therefore, these suboptimal hcs were omitted from further analyses, reducing the final data set from reads to most informative hcs. out of the known msvs within the amplicons, were found to be polymorphic. for rs, exclusively the t allele was observed. in addition, we confirmed in amplicon f of c na and na an msv at position downstream of defb not annotated in dbsnp but present in the hg reference sequence as a paralogous sequence variation. moreover, we identified a novel base exchange in the same amplicon of na located nucleotides further downstream. the msv combinations in out of amplicons could be unambiguously deduced from the overlapping forward and reverse reads. however, for f the read length of the flx chemistry was too short to directly infer complete haplotypes, as the forward reads cover msvs 6- and the reverse reads 7-10a, respectively. nevertheless, using data from non-polymorphic samples, the hc ratios in polymorphic samples and a parsimony assumption, forward and reverse haplotypes collapse consistently into complete f haplotypes. #: number of hcs; *: alleles in lower cases refer to the additionally identified msvs 9a and 10a; (): nominally below 10% threshold; nd: no data. in total, haplotypes could be identified. the largest variety was found for amplicon f with haplotypes out of theoretically possible combinations of msvs. in contrast, only haplotypes were observed for f and f, respectively. the most abundant haplotypes are for f2: ga, f4: tac, f5: tccgggc, f6: cctcg, f7: tcg and f8: ctaccg,. tccgggc and tcg were found in all investigated dnas. joining the haplotypes with the most hcs for each dna sample individually creates inferred "long range haplotypes" of which one is present in of samples. remarkably, this inferred haplotype can be generated by just one recombination of physical haplotypes represented by large insert clones from the rpci- human male bac library, sequenced in the course of the human genome project. several additional msv combinations were found which are represented by only few reads compared to the haplotypes described above, indicating either the generation of chimeric, pcr-mediated recombinant molecules from different target copies, misincorporations during the emulsion pcr, sequencing errors or cross-contaminations. in total, out of pcrs and out of sanger pcrs contained this phenomenon. based on the estimation by alternative methods that the maximal defb cn in our sample set is we discarded hcs with fractions < as such artefacts. this led to the exclusion of and hcs from the and sanger datasets, respectively. comparing the haplotyping results for both methods, the same haplotypes were found in 85% except for amplicons f5/s in samples na and na. in both cases rare haplotypes were not found in the sanger reads. due to the low overall number of f sequences for na the hc frequency for the haplotype inferred from msvs 6- is lower < and had to be excluded. in order to finally confirm or reject the questionable haplotypes, we generated additional f amplicons from the dna samples and performed sequencing in 2/16th plate per sample resulting in and sequences, respectively. from these reads more than hcs per sample for msvs 6- and over calls for msvs 7-10a were obtained for each of the dnas, unequivocally confirming the presence of the questionable ctgggac and ccgggac haplotypes, respectively. #: number of hcs; f = hc frequency; *: alleles in lower cases refer to the additionally identified msvs 9a and 10a. determination of relative defb copy numbers by haplotype calls in order to evaluate the applicability of hcs for the measurement of relative defb cns, we calculated the hc ratios for all amplicons with more than one haplotype and inferred the respective minimal cn. for example, in f from c haplotypes were identified with and calls, respectively. this corresponds to a normalized call ratio of 1:: that is essentially 1:3: indicating a minimal cn of altogether, by this approach cn calculations were done. for samples with more than one polymorphic amplicon we computed the average of the separate cns or of their least common multiple. the final minimal cn estimations for the samples range between and. armour: prt and maph/redvr and personal communication; 5prt. pprt; mlpa. ~: rounded; n: multiple of the minimal estimated cn; *: cn estimations based on ultra-deep sequencing; nd: no data; blank: non polymorphic. the defb cns of the samples haplotyped in this study were previously measured by independent experiments using different prts, mlpa and maph/redvr and armour and hollox, personal communication]. for of the dnas, measurement by the different techniques resulted in identical cns. the remaining samples showed disagreements of only one copy, indicating the consistency of the data. therefore, we took the average of these measurements and compared them with cn estimates from the hcs. for samples the haplotype-based estimates were in agreement with cns determined by the alternative methods. for another dna samples, these numbers differed only by one copy. in the case of na the estimated minimal cn was half of the absolute one. for na msv combinations are polymorphic, resulting in cns of, 2n and, respectively. only in this single case, the hc-based minimal cn estimations can not be unambiguously interpreted and considerably deviates from copies per diploid genome, consistently determined by alternative methods. as ultra-deep sequencing of na and na amplicon f provided most consistent cn estimations for samples with high cns, we further validated this approach by performing a third gs flx run with pooled amplicons f f f and f from these dnas. moreover, we included na and na. to avoid false positive hcs resulting from chimera formation early in pcr, we carried out independent reactions with each primer pair and dna sample. accordingly, we lowered the threshold for exclusion of minor haplotype fractions from 10% to 6% to avoid false negative hcs. altogether, we obtained hcs providing on average ~ per amplicon and sample, of which % were excluded as potential chimera, sequencing errors or contaminations. the extraordinary sequencing depth and the lowered filter threshold identified additional haplotypes for na and na which increased the resolution of the cn estimation. ultra-deep sequencing-based cn estimations for samples with high cns tend to be higher than those determined by the established alternative methods. nd: no data; blank: non polymorphic. discussion impaired beta-defensin synthesis has been described in many human diseased states, namely in inflammatory disorders. while association of a disease and defb cn has been demonstrated, associations of sequence variants with disease cannot reliably be investigated with established genotyping methods. hollox observed that it may be the nucleotide state of an msv that actually causes susceptibility to a disease for which association with cn has been found. if so, cn is only a proxy for causative sequence variants. however, description of sequence variation in cn variable loci such as defb at 8p is inherently challenging. currently, neither the haplotype structure of defbs nor their arrangements on chromosomes are known. the present work provides a first glimpse of the defb haplotype complexity through amplicon sequencing of selected polymorphic defensin gene fragments. among the currently available next generation sequencing approaches, the technology is best suited for this purpose as it provides longest reads that in turn produce the longest inferred physical haplotypes. eleven dna samples derived from lcls were selected to represent a range of 2- defb copies per diploid genome. all dnas are publicly available and were investigated in the framework of the hapmap project. four of the lcls were established from individuals of caucasian, of yoruba and one of chinese ancestry. about msv hcs were derived from amplicons containing 2- annotated msvs each. one msv not deposited in dbsnp and a new one were detected in samples of yoruba and one of caucasian ancestry, respectively. theoretically, the msvs in amplicons altogether could compose haplotypes of which we identified. as exemplified by the analysis of the f amplicon data, hc ratios provided by the deep sequencing approach may serve as additional experimental evidence to deduce longer haplotypes than the maximal ngs read length. with msvs in amplicons, totalling to bp, the msv density in the investigated regions is approximately msv/kb. this is higher than the overall single nucleotide variation density of the entire defb currently annotated in dbsnp and reflects the directed selection of the amplified regions to screen a maximum of polymorphic positions within a short distance. in comparison, for the most sequence variable locus of the human genome, the mhc complex, alignments of mhc haplotypes revealed snp/kb with a higher density in the intragenic regions. for the genomic loci of hla-a, b and c there are polymorphic positions within bp assigned by dbsnp build corresponding to snps/kb. this indicates that in terms of sequence variability, defb is substantially less polymorphic than the hla loci, and that defb together with the analysed amplicons are comparable to the mhc intragenic regions. the fact that the deep-sequencing revealed only one novel msv on the background of previously known ones shows that the dbsnp annotation of the analysed regions is quite comprehensive. on the other hand according to dbsnp, the msv density of defb is about times higher than the snp density of the entire human genome. moreover, currently unknown additional sequence variability may be still hidden in the defb which may be revealed by future targeted and whole-genome resequencing efforts. the haplotypes identified by sequencing and by cloning/sanger sequencing were identical except for cases, which could be resolved by additional sequencing. however, the ratios of haplotypes derived from a given individual amplicon sometimes differ remarkably. for example, the c f haplotypes ccggac and cccggc were revealed by sequencing in a ratio of 1: but 1: by sanger derived reads. we suspect the explanation is that the number of hcs from sanger sequenced clones is too low for providing reliable results. moreover, additional bias may be introduced e.g. by the bacterial cloning step. theoretically, the more hcs that are available, the more accurately they should reflect the real ratio of the defb copies with particular haplotypes. this becomes more important as the number of defbs and/or haplotypes per genome increases. the conclusion is supported by our efforts to resolve the discrepancies between and sanger sequencing for na and na the minor haplotypes of the f amplicon were supported by but not by sanger sequencing, probably due to the low amount of sequenced clones. additional hcs finally confirmed the presence of the f haplotypes under question. furthermore we noticed an uneven distribution of forward and reverse reads in the sequencing of particular amplicons. this is obvious in fig. with a nearly 1: forward-to-reverse read ratio and is also reflected in the entire data set of amplicon f with forward but reverse hcs. such a skew may be caused by unequal sequencing efficiencies due to the direction-specific sequencing primers as well as to differences in base composition and/or motifs between the forward and reverse strands. thus in the case of non or partially overlapping forward and reverse reads, the resolution of the approach is limited by the less effective sequencing direction. another problem of any pcr-based haplotyping is the occurrence of chimeric products. since both amplicon sequencing and the cloning/sanger approach are based on pcr, the formation of such artefacts must be taken into account. chimeras can be generated during pcr by heteroduplexes of e.g. a complete copy of one allele with a fragment from another allele. completion of the partial strand in the elongation step creates chimeric molecules from which chimeric haplotypes may be erroneously determined. the degree of chimera formation is dependent on various factors such as polymerase, nucleotide/primer concentrations, cycling conditions and amplicon length. in addition to the formation of chimeric pcr products, nucleotide misincorporations during the emulsion pcr, sequencing errors and cross-contaminations have to be taken into account. in the current study, we have appointed and discarded an msv combination as artificial if its relative number of call was lower than 10% or 6%. this led to the exclusion of less than 3% of all hcs which otherwise would render the analysis impossible. false negative hcs by this cutoff per definitionem would only affect dna samples with more than and defb copies, respectively. cns > are rarely reported in the literature. nevertheless, these threshold filters may fail if the artificial molecule appears early in pcr. to avoid respective misinterpretations, independent pcrs were carried out and pooled before sequencing for the third gs flx run. the estimation of cns from the hc ratios is intrinsically problematic, since ratios provide only minimal cns and any multiple of that can be ruled out only by additional considerations. moreover, slightly different ratios lead to different cn estimations. nevertheless, thanks to the high number of ngs hcs, cns could be correctly determined for out of samples and differed only by one copy per diploid genome for another samples. remarkably, the high hc numbers obtained by ultra-deep gs flx runs and consistently point towards higher cns than obtained by the widely used prts/mpla. it is known that the latter methods tend to underestimate the actual cn in case of defb cns >. thus, it has to be proven in the future, whether ultra-deep ngs like may provide more reliable results than the currently established methods for cases of high cns per diploid genome. the recently developed mrfast points in the same direction although the sequencing depth of personalized whole-genome sequencing efforts and the genomes project is too low to provide sufficient read counts for reliable estimations of high cns in complex regions like defb. only in one case were the cn estimations inconsistent. for this, we suppose possible reasons. first, the estimated cn results from amplicon f with a relatively low amount of reads which in case of the forward sequencing direction caused a random deviation from the 1: ratio observed for the reverse hcs. second, 7- copies were calculated by the ratio of haplotypes in amplicon f as the haplotypes were confirmed by ultra-deep sequencing of pooled independent amplicons, it is unlikely that the minor haplotype is a chimera. as the remaining amplicons show no sequence variation and are therefore in agreement with the low cn of determined by the alternative methods, sample na may represent in our hands the first example violating the concordance rule of defb. whether the underlying molecular event represents a natural polymorphism or emerged in cell culture remains to be elucidated in the future. although our results indicate that ngs based haplotyping and cn estimation thereof is a suitable approach to characterize a highly polymorphic and cn variable loci as the defb cluster, this method has some inherent limitations. first, haplotyping is restricted to those regions in which at least two msvs are located within the read length of the applied ngs technology. second, equimolar pooling of pcr products is difficult and also the efficiencies of the following emulsion pcr may deviate among the amplicons. this can result in differing amounts of hcs per amplicon which may prevent reliable haplotyping if too few sequences are obtained. third, formation of pcr artefacts leading to the identification of "false", i.e. chimeric haplotypes always has to be considered. although distinction between "true" and "false" halpotypes is easier with higher sequencing depth it is problematic for samples with high cns.  deep amplicon sequencing is an effective method for parallel haplotyping and cn estimation in highly polymorphic loci such as defb at chromosome 8p for the following reasons: there is no bacterial cloning bias as a source of putative errors in haplotype identification. moreover, in contrast to the cloning/sanger sequencing approach, thousands of hcs per amplicon can easily be performed leading to a higher accuracy in obtained haplotype ratios and cn estimations inferred thereof. calculated on the basis of the consumable's prices for the present study and normalized to the same yield of informative hcs, the costs for amplicon sequencing amounts to only ~4% of those for cloning/sanger sequencing. although this does not even consider the reduced experimental effort it is still too expensive for high-throughput measurements but acceptable for analyses of small cohorts. recently, titanium amplicon sequencing protocols and kits became available, increasing achievable read lengths to bp with a 2-fold increase of the number of reads per run. thus, longer amplicons than in our study can be sequenced, encompassing more or more distant msvs. moreover, as the output of reads also increases, multiplexing different dnas will be feasible by application of barcoding strategies. although the presented amplicon sequencing delivers only relative cns and has to be completed by other approaches, future inclusion of reference amplicons from loci with invariable cn will provide absolute defb cns. deep amplicon sequencing can contribute substantially to investigate sequence variants and haplotype structure of cn variable loci. nevertheless, increases in both ngs read length and output are needed to eventually completely resolve loci like defb at the physical haplotype level. authors' contributions st designed the experiments and carried out both the and sanger experiments, designed by kh. mg performed the mlpa and prt experiments. st, ap and ks analyzed the data. mp, st, jh, pr and ss conceived of the study, and participated in its design and coordination. st, mp, kh and ks wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 amplicons, primers and their locations in the human genome. pcr products, primers used for their amplification and chromosomal locations of the amplified regions. msvs and amplicons. analyzed multisite variations, their rs numbers and chromosomal locations according to snpdb build and ncbi build, respectively. numbers of assembled gs-flx reads per amplicon and dna. assembled sequences from gs flx runs per dna and amplicon. haplotypes and haplotype calls for gs-flx runs 1- and pcr/cloning. haplotypes, haplotype calls and their fraction per dna and msv combination, derived from gs flx runs and and sanger sequencing of cloned pcr products. haplotypes by msv combinations. haplotypes and total number and fraction of haplotype summed up from all dnas sequenced in gs flx runs and haplotypes by dna. identified haplotypes per dna and amplicon, highlighting the most abundant haplotypes. individual defb copy numbers derived from the ratio of haplotype calls per msv combination and comparison with cns by other methods. estimation of of the defb cluster copy number from the ratio of haplotype calls per dna and amplicon and comparison of with cns determined by mlpa and different paralogue ratio tests. haplotypes and haplotype calls for gs-flx run haplotypes, haplotype calls and their fraction per dna and msv combination, derived from gs flx run. individual defb copy numbers derived from the ratio of haplotype calls per msv combination from run1+ versus run individual defb copy numbers for dnas in comparison between gs flx run 1+ versus gs flx run. click here for file acknowledgements we thank daniela werler, cornelia luge, silke foerste and beate szafranski for excellent technical assistance. john armour and ed hollox are acknowledged for kindly providing cn data based on maph/redvr and prt. we thank niels jahn for bioinformatics support as well as roger horton and andrew heidel for critically reading the manuscript. this work was supported by grants from the bundesministerium für bildung und forschung and the gp project of the network of environmental disorders.
minimal cns were estimated from the ratio of hcs and compared to absolute cns determined by alternative methods. the beta-defensin gene cluster at chromosome 8p is one of the most copy number variable regions of the human genome. for samples and amplicons, the haplotyping results were compared to those by cloning/sanger sequencing. whereas individual defb cns have been suggested as independent genetic risk factors for several diseases, the role of multisite sequence variations is less well understood and to date has only been reported for prostate cancer. concordance in cns was found for samples, the cns differed by one in samples and the estimated minimal cn was half of the absolute in one sample. intrinsic problems related to chimera formation during pcr and differences between haplotyping by and cloning/sanger sequencing are discussed. deep amplicon sequencing using the technology yield thousands of hcs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and cn estimation in small to medium-sized cohorts. here, we demonstrate that amplicon sequencing of pooled individual pcr products by the technology allows in-depth determination of msv haplotypes and estimation of defb cns in parallel. the obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly cn variable loci such as the beta-defensin locus. six pcr products spread over ~ kb of defb and harbouring known msvs were amplified from dna samples, pooled and sequenced on a roche gs flx sequencer.
 the beta-defensin gene cluster at chromosome 8p is one of the most copy number variable regions of the human genome. whereas individual defb cns have been suggested as independent genetic risk factors for several diseases, the role of multisite sequence variations is less well understood and to date has only been reported for prostate cancer. simultaneous assessment of msvs and cns can be achieved by pcr, cloning and sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. here, we demonstrate that amplicon sequencing of pooled individual pcr products by the technology allows in-depth determination of msv haplotypes and estimation of defb cns in parallel.  six pcr products spread over ~ kb of defb and harbouring known msvs were amplified from dna samples, pooled and sequenced on a roche gs flx sequencer. from ~ reads, ~ haplotype calls were inferred that identified haplotypes ranging from to per amplicon. in addition to the known msvs, two additional sequence variations were detected. minimal cns were estimated from the ratio of hcs and compared to absolute cns determined by alternative methods. concordance in cns was found for samples, the cns differed by one in samples and the estimated minimal cn was half of the absolute in one sample. for samples and amplicons, the haplotyping results were compared to those by cloning/sanger sequencing. intrinsic problems related to chimera formation during pcr and differences between haplotyping by and cloning/sanger sequencing are discussed.  deep amplicon sequencing using the technology yield thousands of hcs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and cn estimation in small to medium-sized cohorts. the obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly cn variable loci such as the beta-defensin locus. 