BACKGROUND
mobile elements  are dna sequences that can replicate and insert themselves into new loci within larger host genomes.
this strategy has proved very successful: mes are evolutionarily ancient, highly diversified in form, ubiquitous in distribution, and often extremely numerous within their host populations  <cit> .
whole genome sequencing of representatives of many species has allowed great insight into the diversity, number and deep evolutionary history of mes  <cit> , but it provides very limited information about the frequencies and distributions of me insertions in populations.
much of our knowledge about the patterns of me insertion variation, especially in humans, has been collected by first ascertaining polymorphic me insertion loci in a small sample of chromosomes and then genotyping those loci in a larger sample from the population .
in some cases, loci that contain insertions of recently-active mes are identified in publicly available dna sequences , then screened by pcr and gel electrophoresis in a small number of individuals to identify the loci that are polymorphic .
in other cases, polymorphic loci are identified by comparing publicly available dna sequences from multiple individuals .
the pcr-based locus-by-locus genotyping approach is labor intensive when many loci and individuals are being studied.
moreover, these approaches are limited to identifying and studying polymorphisms that are common in the populations and genomic regions that are best represented in public sequence databases, which introduces ascertainment biases into the data.
since dna sequences in public databases are collected by heterogeneous methods for various purposes, it is difficult to quantitatively model those ascertainment biases, and investigations that require specific sampling designs  are precluded.
current full-genome sequencing methods remain prohibitively expensive for studies of population variation, and it is difficult to reliably identify indels of any kind in low-coverage genomes assembled from short reads.
methods that rely on subtractive hybridization  <cit>  allow researchers to efficiently ascertain polymorphic insertion loci in population samples of their own design, but the number of loci that can be identified using these methods has been limited by the number of samples that can be processed simultaneously and the effort required to confirm candidate loci by cloning and sequencing.
transposon display  methods  <cit>  can generate presence/absence genotypes for a subset of me insertion loci in all members of a sample.
td avoids ascertainment bias for common insertions, since the specific subset of loci that are genotyped is determined by the choice of restriction enzymes and design of pcr primers, not by the frequencies of the insertions.
the number of loci that can be genotyped at one time is limited by the requirement that each insertion be identifiable as a unique, reliably distinguishable band on a polyacrylamide gel.
since insertions at both fixed and variable loci generate bands, large numbers of fixed "background" insertions  can greatly reduce the useful genotyping capacity of a td experiment.
in order to confirm results or compare them across methods, it is typically necessary to dissect pcr products out of bands on td gels for reamplification, cloning and sequencing.
here we describe a cost-effective method for accurately and quickly identifying nearly all insertions of a given mobile element family in every individual of a large sample.
mobile element scanning  relies on targeted high-throughput sequencing to efficiently read nearly all the junctions between insertions of a class of mobile elements and the genomic flanks of those insertions.
high target specificity  is achieved by using element-specific pcr and sequencing primers to amplify and sequence only the desired element-flank junctions.
each mobile element insertion is uniquely identified by its precise insertion position and the flanking genomic sequence, and the presence or absence of that junction sequence in sequence reads derived from an individual dna sample indicates the presence or absence of the insertion in that individual.
to make efficient use of the capacity of high-throughput sequencing platforms, each individual sample is labeled with a unique 5-bp index, and multiple samples are pooled together for sequencing .
the aluyb <dig> and aluyb <dig> retrotransposon subfamilies  in the human genome provide an interesting and challenging target for testing me-scan.
due to the recent retrotranspositional activity of the aluyb8/ <dig> subfamily, there are thousands of insertion loci to be assayed, including many polymorphic ones  <cit> .
the long history of alu retrotransposition in primates has created a background of nearly one million older alu copies  <cit>  that must be avoided in order to assay aluyb8/ <dig> insertions alone.
finally, the human genome reference sequence  <cit>  and previous alu-genotyping studies  <cit>  provide independent information against which we can measure the performance of me-scan.
in this study, we demonstrate that me-scan makes efficient use of sequencing output; that pooled libraries are evenly represented in the sequencing results; and that we are able to identify aluyb8/ <dig> insertions with a sensitivity and specificity of ~95%
RESULTS
overview of me-scan
the key steps of the me-scan protocol are illustrated in figure  <dig>  described below, and detailed in methods.
it combines and adapts established methods of transposon display  <cit> , high-throughput sequencing  <cit> , and sample indexing  <cit> .
genomic dna  is sheared by sonication.
the resulting double-stranded dna fragments are repaired to blunt ends, and unpaired adenosines are added to the 3' ends.
oligonucleotide adapters with an unpaired 3' thymine are ligated to those fragments .
the adapters contain a 5-bp "index" sequence that is unique to each individual sample.
this index is later sequenced and links each read pair with the individual from whom it is derived, so samples can be pooled  for subsequent steps.
at this point, every sequence from every individual's genome is represented by many dsdna fragments.
to extract only those fragments that contain an aluyb8/ <dig>  we perform pcr with a biotinylated primer that anneals to a site found only in aluyb8/ <dig> elements and a primer that matches the adapter sequence .
this pcr generates some molecules that contain only a fragment of an alu element and no flanking genomic sequence.
size selection by gel electrophoresis is used to enrich the resulting pcr product for molecules that contain most of an alu insertion as well as 50- <dig> bp of upstream flanking genomic sequence.
the biotinylated strands are then bound to streptavidin-coated paramagnetic beads and purified away from nonspecific dna by magnetic separation .
the product is re-amplified and checked for quality and quantity .
the resulting aluyb8-specific dna fragment library is subjected to paired-end sequencing  on one flow cell lane of an illumina genome analyzer ii .
instead of using illumina's standard primer for the first sequencing read, we use a custom sequencing primer that anneals to a site  <dig> bp inside the 5' end of the consensus aluyb8/ <dig> sequence.
thus the first read  will typically consist of  <dig> bp of aluyb8/ <dig> sequence followed by  <dig> bp of genomic sequence.
the second-end read  extends from illumina's standard primer and will contain a 5-bp individual-specific index, a 't', and  <dig> bp of genomic sequence.
application of me-scan to human aluyb8/ <dig> elements
yield of usable sequence data
*supports one of the  <dig>  insertion loci reported in additional file  <dig>
experimental design
we designed two sequencing experiments to test the reproducibility, specificity and sensitivity of me-scan at various levels of sequencing coverage .
dna samples from four individuals  were used.
for the "replication" experiment, we processed two aliquots of dna from individual a in parallel through the ligation step, using a different index for each aliquot, then pooled them in equal amounts for subsequent library preparation and sequencing.
for the "pooling" experiment, we prepared five samples with different indexes  and combined them in varying proportions  to construct a pooled library.
figure  <dig> shows that the proportions of each index as recovered by sequencing match the intended proportions to within 1% on average .
thus, it should be possible to pool small amounts of dna from many library preparations without severely under- or over-representing any one library.
* only read pairs passing all qc filters are counted.
<dig> two aliquots of a dna sample from this individual were processed in parallel and pooled for sequencing.
<dig> a dna sample from this individual was sonicated, then divided into two aliquots for subsequent library construction steps.
sensitivity and specificity
to estimate the sensitivity of me-scan, we searched for false negative results in our data.
we first identified a set of presumably fixed aluyb8/ <dig> insertions, reasoning that me-scan should retrieve all of these insertions in every individual, and that failures to do so would likely represent false negative errors.
these insertions are present in the human reference genome assembly, contain good matches to the aluyb8/9-specific amplification and sequencing primers we used , are of the canonical aluyb8/ <dig> size , and are not known to be polymorphic .
we found  <dig>  such loci .
the numbers of these loci that were not retrieved in each sample preparation are shown in table  <dig>  on the assumption that each of these  <dig>  loci is indeed fixed for the insertion in humans, the rate of me-scan false negative results ranges from 8% to 25%.
the error rate depends on the sequencing effort expended on a sample: the higher the number of reads, the lower the false negative rate.
<dig> the number of genotypes compared differs from sample to sample due to occasional uncertain genotype calls  in the pcr and gel genotyping assays.
<dig> these genotypes represent actual false negative me-scan results
<dig> for  <dig> of the  <dig>  loci, me-scan did not observe the insertion-present allele in any of the samples tested .
out of this set of  <dig>  insertion loci, there were  <dig> loci for which the insertion-present state was not observed by me-scan in any of our four samples .
thirty-three of these  <dig>  are part of recently duplicated regions of the genome .
read pairs derived from these do not map to a unique position in the genome, and are therefore eliminated by our analysis pipeline.
read pairs corresponding to previously unknown alu insertions into such regions would likewise be eliminated and lead to false negative results.
another  <dig> of those  <dig> insertions lack detectable alu sequence in the  <dig> bp immediately following the sequencing primer binding site.
nearly all of these insertions are in fact observed in the me-scan sequencing results.
however, in order to filter out artifactual false positives, the me-scan pipeline discards loci that lack the expected alu sequence, and so these loci lead to false negative results.
apart from the loci in recently duplicated regions and those lacking the expected alu sequence in the junction read, it is very likely that some of the  <dig>  putatively fixed aluyb8/ <dig> elements are in fact not fixed, and are absent in some of our samples.
if so, their absences from the me-scan results do not represent false negatives.
we used pcr and gel electrophoresis to genotype  <dig> loci that were not in duplicated regions and contained the expected alu sequence in the junction read, but still were not retrieved by me-scan in some or all of our samples .
the genotypes obtained by pcr and gel-typing for the four individuals are compared with those obtained by me-scan in table  <dig>  in the current me-scan analysis pipeline, alu insertions are treated as presence or absence states for purposes of comparison.
of the  <dig> loci examined, only one had the insertion present in the homozygous state in all four individuals.
four more loci had the insertion present in at least some individuals, but the remaining  <dig> loci were homozygous for the absence of the insertion in all individuals.
initially, our sensitivity appears to be ~90% at a sequencing coverage of ~ <dig>  read pairs per sample  pcr genotyping suggests that only ~12%  of the  <dig> putative false negative genotypes that were not in recently duplicated regions are in fact false negatives, so a better estimate of the number of actual false negative results in this experiment is  <dig> , which implies a sensitivity of  <dig> %.
if we restrict our target set of alu insertions to only those that have intact 5' ends and reside in non-duplicated regions, our sensitivity exceeds 99%.
to estimate the specificity of me-scan, we searched for examples of false positive results.
any false positives should be in the set of new aluyb8/ <dig> insertions retrieved by me-scan, i.e., those that we could not annotate based on the reference genome  we identified  <dig> such insertions.
we selected  <dig> loci at random from this set for confirmation by pcr and gel electrophoresis .
all  <dig> showed the pcr product expected from the alu-filled allele in at least one sample, indicating that the method is very reliable for detecting new insertion loci.
in some individuals, however, an insertion detected by me-scan was not detected by pcr and gel electrophoresis, suggesting either that the gel-typing assay was insufficiently sensitive or that there was some cross-contamination between me-scan libraries.
on the assumption that the me-scan results are at fault, the false-positive rate ranged from 3% in the highest-coverage samples to 10% in the lowest-coverage samples .
the combined biochemical and bioinformatic protocols achieve a specificity of ~96% when based on  <dig>  reads per sample  because all individuals were genotyped by me-scan and pcr plus gel electrophoresis for these  <dig> loci, this data set allows us to examine the rate of false negative me-scan results for this class of loci .
no false negatives were observed in the four higher-coverage samples, suggesting that the false negative rate among new alu insertions is as low as that observed for known aluyb8/ <dig> loci  the data for the three low-coverage samples suggest a higher false-negative rate among new alu insertion loci when relying on lower-coverage sequencing.
* not observed in the human reference genome .
confirmation of new variable aluyb8/ <dig> insertion loci in additional individuals
in order to test the dimorphic status of loci in the above-described set of  <dig> new aluyb8/ <dig> insertions, we genotyped them by pcr  in a panel of eight additional unrelated individuals: six of east asian descent , one european , and one african individual .
the genotyping results are reported in additional file  <dig>  of the  <dig> loci,  <dig> showed the expected alu insertion in at least one individual, confirming that they are dimorphic in humans.
of the loci that lacked the insertion allele in this panel of eight individuals, one did not show the insertion even in the original panel of four individuals , and three showed the insertion in only one individual of the four.
any comprehensive survey of genetic variation will identify "singletons" , so our results are consistent with that expectation.
these three insertions could be rare in humans, they could be limited to a family or just a single individual , or they might be non-heritable insertions resulting from somatic or even cell-line retrotransposition events.
in the full set of  <dig> new aluyb8/ <dig> insertions identified by me-scan ,  <dig> were observed  in more than one individual of the original four, which implies that they are polymorphic in humans.
the remaining  <dig> insertions could be rare, de novo, or even non-germline insertions.
however,  <dig> of those  <dig> loci were among the  <dig> chosen for further genotyping in the panel of  <dig> additional individuals, and of those  <dig> loci,  <dig>  had the insertion present in at least one member of the panel.
thus it is likely that a large majority of the  <dig> new aluyb8/ <dig> loci identified by me-scan are in fact polymorphic in humans.
reproducibility
in order to study the reproducibility of me-scan, we assayed dna from individual a twice, in parallel and with the same level of coverage  table  <dig> shows the results of comparing the alu insertions identified by me-scan in the two replicates.
together, the two replicates identified  <dig>  aluyb8/ <dig> insertions that are known from the reference genome  among these, the proportion of insertions that identified in one replicate but not the other  is less than 1%.
these replicates also recovered  <dig>  previously known insertions from alu families other than aluyb8/ <dig>  but with low reproducibility: each replicate missed more than  <dig> of the insertions identified in the other.
the genome contains hundreds of thousands of alu insertions with poor matches to the amplification and sequencing primers we used, so despite the low probability that any one of them will be amplified and sequenced, we expect to see sporadic amplification of some of these loci in each experiment.
by the same token, however, the set of loci that are sporadically amplified will differ from sample to sample, so their reproducibility should be low.
the two replicates also yielded  <dig> new alu insertions , with a replication rate of ~92% in that set .
this is slightly lower than the ~96% sensitivity observed for the set of known insertions  perhaps this set of previously unobserved alu insertion loci includes some from other currently active alu families, such as aluya <dig>  these loci may amplify erratically due to their poorly matching primer binding sites, thereby reducing the overall reliability in this set.
* total positive loci are those observed in either of the two samples of the 'replication' experiment, indexed accat and tattc.
† number of insertion loci absent in a sample, divided by the total observed in both samples, averaged over the two samples.
comparison with previously genotyped aluyb8/ <dig> insertion loci
numerous researchers, including ourselves, have studied polymorphic alu insertion loci by first ascertaining them in a small set of individuals and then genotyping them in a larger set using locus-specific pcr primers and gel electrophoresis  <cit> .
we previously used this method to genotype  <dig> polymorphic aluyb8/ <dig> loci in the four individuals that we assayed by me-scan in the current work  <cit>  .
table  <dig> shows the results of comparing the genotypes inferred by both methods for those loci in these individuals.
most of the disagreements between the two methods  are due to negative me-scan genotypes in the low-coverage samples  it seems likely that these gel-typing results are correct, and that these disagreements are me-scan false negatives due to insufficient sequencing depth.
the remaining four disagreements involve three replicate me-scan genotypes of a single locus in one individual  and one genotype at another locus in another individual .
me-scan indicates the presence of an insertion in these two cases, while previous genotyping indicated homozygous absent genotypes.
the consistency of the me-scan result for the one locus in individual a suggests that the previous genotyping results might be at fault, so we used pcr and gel electrophoresis to check both genotypes  we find that, in fact, both genotypes were heterozygous for the presence of the insertion, in agreement with the me-scan results  for this set of loci, given sufficient coverage , me-scan appears to be more accurate than gel typing.
* all four of the apparent me-scan false positives are due to errors  in the previous pcr- and gel-based genotyping.
the huref genome sequence of j. craig venter  <cit>  offers the opportunity to verify some of the alu insertions we identified that are absent from the hg19/grch <dig> reference.
of  <dig> huref aluyb <dig> and aluyb <dig> insertions of at least  <dig> bp in length  <cit> ,  <dig> were observed by me-scan .
although we have no precise expectation for the number of aluyb8/ <dig> insertions that our panel of four east asian individuals should share with one european , this result is the equivalent of confirmation by complete sequencing of a sizable set of the previously unobserved loci identified by me-scan.
discussion
complete ascertainment and genotyping of alu insertion events in large population samples would give us - for the first time - a clear and panoramic view of their population dynamics.
knowing the genomic distribution of the most recent alu insertions will allow us to decisively disentangle the effects of target site preferences from the effects of natural selection, recombination, and gene conversion that may subsequently alter that distribution  <cit> .
from the full site frequency spectrum of insertions, we should be able to estimate the transposition rate and the range of selection coefficients that affect alu insertions  <cit> .
it may even be possible to identify factors that cause positive or negative selection on some insertion classes.
with sufficient data, we may be able to estimate the evolutionarily relevant, in vivo transpositional activities and effects of specific alu elements  by analyzing correlated variation in transposition rates between families and populations.
we have developed a method for quickly, sensitively, and specifically ascertaining nearly all aluyb8/ <dig> insertions in several individual samples simultaneously.
other researchers have used similar targeted high-throughput sequencing approaches to identify me insertions  <cit> , but with very different goals.
typically, an engineered me is used to create many highly uniform insertions of a unique dna construct in a model organism or cell line.
selection for a phenotype is then applied, and targeted high-throughput sequencing is used to locate a sample of the selected insertions.
me-scan is designed to identify essentially all members of a class of naturally occurring insertions against a large background of very similar sequences in all members of a population sample.
by using a fraction of the sequencing capacity of two illumina gaii flow cell lanes to scan four individuals, we have simultaneously identified, mapped, and partially genotyped  <dig> new variable aluyb8/ <dig> insertions in those individuals.
nearly all previously known aluyb8/ <dig> insertion loci were also detected.
we examined several levels of sequencing effort and found that a level of ~ <dig>  read pairs per sample allowed the assay to achieve ~95% sensitivity and specificity.
illumina's genome analyzer iix now yields up to  <dig> million paired sequence reads per flow cell lane  <cit> .
if this read density is approachable with me-scan libraries, it will be possible to index and pool  <dig> individuals per lane with very high sensitivity and specificity.
even at lower read densities, it should be possible to make more efficient use of the sequencing yield.
theoretically, the number of read pairs per insertion should be poisson-distributed, with variance equal to the mean coverage.
however, in all our samples, the variances are at least an order of magnitude greater than the means.
this is a common feature of high-throughput fragment-library sequencing techniques .
reducing this overdispersion even moderately would deliver higher performance at lower levels of sequencing effort by reducing the number of read pairs that are effectively wasted on heavily over-represented insertions.
it would also increase the number of reads derived from poorly represented insertions that are at risk of being missed altogether.
simple changes to the basic protocol  are likely to reduce that overdispersion.
the length of sequencing reads has increased along with the number of reads per run.
it is now possible to obtain paired  <dig> bp sequence reads instead of the paired  <dig> bp reads used here  <cit> .
longer reads would increase both the sensitivity and specificity of the me-scan analysis pipeline.
by using a sequencing primer that anneals ~ <dig> bp inside the normal 5' end of an alu  and reading  <dig> bp from that primer, one could obtain more of an alu insertion's sequence  as well as more immediately adjacent genomic sequence .
the additional genomic sequence would allow reliable mapping of some read pairs that are now being discarded .
the additional alu sequence would allow the elimination of spurious non-alu products without discarding evidence of alu insertions with modest 5' truncations.
a short second read would still be required to sequence the sample index.
more importantly, using a long  junction read would allow construction of a reference-independent analysis pipeline.
the me-scan analysis pipeline described above depends on mapping reads to a reference genome for two reasons.
firstly, most of our analyses focused on comparing me-scan results to the "gold standard" information in the reference genome, so mapping was required in any case.
secondly, the information in paired  <dig> bp reads is insufficient for highly accurate reference-independent locus identification and genotyping.
the first  <dig> bp of an alu junction read is usually alu sequence, which - being nearly identical for most alu elements - does not distinguish groups of read pairs that represent different insertion loci.
the remaining  <dig> bp  are often insufficient to clearly distinguish all the groups of reads, since some ambiguity must be allowed to handle sequencing errors and some insertions are in regions of low sequence complexity.
when mapping read pairs to a reference, those  <dig> bp combine with the  <dig> bp of genomic sequence from the flank read to provide enough information for reliable positioning.
this allows read pairs to be grouped according to their mapped location.
unfortunately, since the  <dig> bp segments of genomic sequence in the flank reads from a particular insertion are derived from sites at varying distances from the junction read, they will not be identical and may not even overlap.
consequently, the flank reads cannot be used to reliably group read pairs based on their sequence alone.
however, with a long alu junction read , it should be possible to identify and group all junction reads that represent a particular insertion simply because they will all share nearly identical junction sequences, while reads from different loci will almost always be easily distinguished due to their different sequences  it would no longer be necessary to first map every read pair to a reference genome sequence and then group them into loci by their positions.
genotyping by grouping read pairs according to the near-identity of their junction sequences should deliver accuracy as good as or better than what we have achieved here.
in particular, new insertions into one copy of a duplicated genomic segment will not pose a problem.
the position of the new insertion within the duplication will be unique, and all reads corresponding to that insertion will group together, just as they would for new insertions into unique regions.
the ability to ascertain and genotype me insertions without relying on a sequenced reference genome would make me-scan very widely applicable: by designing the appropriate amplification and sequencing primers, one could study the population dynamics of nearly any me family in any organism in detail.
the method promises to be robust, quick, reliable and relatively inexpensive: the per-bp cost of sequencing continues to decrease; the indexing and pooling method makes efficient use of high-capacity sequencing platforms; library preparation is technically straightforward; the cost of sample preparation is minimized by the use of sonication rather than nebulization, and by pooling many samples for some processing steps; and the amount of template required has decreased by at least fivefold since we began our experiments  <cit> , further reducing the cost of reagents required for sample preparation.
these factors would make a reference-independent me-scan method appealing for simultaneously identifying and genotyping polymorphic markers of population genetic diversity in any species.
CONCLUSIONS
me-scan enables the rapid, efficient, and highly accurate ascertainment and genotyping  of thousands of mobile element insertion loci of a particular subfamily in many individuals, even in the presence of a very large background of related elements.
with longer sequencing read lengths, it will be possible to apply me-scan to species for which no full-genome reference sequence is available.
the ability to quickly and inexpensively gather so much detailed information on any me family in any population represents a breakthrough in the study of the population dynamics and evolution of mobile elements.

 mobile elements are dna sequences that can replicate and insert themselves into new loci within larger host genomes. this strategy has proved very successful: mes are evolutionarily ancient, highly diversified in form, ubiquitous in distribution, and often extremely numerous within their host populations. whole genome sequencing of representatives of many species has allowed great insight into the diversity, number and deep evolutionary history of mes, but it provides very limited information about the frequencies and distributions of me insertions in populations. much of our knowledge about the patterns of me insertion variation, especially in humans, has been collected by first ascertaining polymorphic me insertion loci in a small sample of chromosomes and then genotyping those loci in a larger sample from the population. in some cases, loci that contain insertions of recently-active mes are identified in publicly available dna sequences, then screened by pcr and gel electrophoresis in a small number of individuals to identify the loci that are polymorphic. in other cases, polymorphic loci are identified by comparing publicly available dna sequences from multiple individuals. the pcr-based locus-by-locus genotyping approach is labor intensive when many loci and individuals are being studied. moreover, these approaches are limited to identifying and studying polymorphisms that are common in the populations and genomic regions that are best represented in public sequence databases, which introduces ascertainment biases into the data. since dna sequences in public databases are collected by heterogeneous methods for various purposes, it is difficult to quantitatively model those ascertainment biases, and investigations that require specific sampling designs are precluded. current full-genome sequencing methods remain prohibitively expensive for studies of population variation, and it is difficult to reliably identify indels of any kind in low-coverage genomes assembled from short reads. methods that rely on subtractive hybridization allow researchers to efficiently ascertain polymorphic insertion loci in population samples of their own design, but the number of loci that can be identified using these methods has been limited by the number of samples that can be processed simultaneously and the effort required to confirm candidate loci by cloning and sequencing. transposon display methods can generate presence/absence genotypes for a subset of me insertion loci in all members of a sample. td avoids ascertainment bias for common insertions, since the specific subset of loci that are genotyped is determined by the choice of restriction enzymes and design of pcr primers, not by the frequencies of the insertions. the number of loci that can be genotyped at one time is limited by the requirement that each insertion be identifiable as a unique, reliably distinguishable band on a polyacrylamide gel. since insertions at both fixed and variable loci generate bands, large numbers of fixed "background" insertions can greatly reduce the useful genotyping capacity of a td experiment. in order to confirm results or compare them across methods, it is typically necessary to dissect pcr products out of bands on td gels for reamplification, cloning and sequencing. here we describe a cost-effective method for accurately and quickly identifying nearly all insertions of a given mobile element family in every individual of a large sample. mobile element scanning relies on targeted high-throughput sequencing to efficiently read nearly all the junctions between insertions of a class of mobile elements and the genomic flanks of those insertions. high target specificity is achieved by using element-specific pcr and sequencing primers to amplify and sequence only the desired element-flank junctions. each mobile element insertion is uniquely identified by its precise insertion position and the flanking genomic sequence, and the presence or absence of that junction sequence in sequence reads derived from an individual dna sample indicates the presence or absence of the insertion in that individual. to make efficient use of the capacity of high-throughput sequencing platforms, each individual sample is labeled with a unique 5-bp index, and multiple samples are pooled together for sequencing. the aluyb and aluyb retrotransposon subfamilies in the human genome provide an interesting and challenging target for testing me-scan. due to the recent retrotranspositional activity of the aluyb8/ subfamily, there are thousands of insertion loci to be assayed, including many polymorphic ones. the long history of alu retrotransposition in primates has created a background of nearly one million older alu copies that must be avoided in order to assay aluyb8/ insertions alone. finally, the human genome reference sequence and previous alu-genotyping studies provide independent information against which we can measure the performance of me-scan. in this study, we demonstrate that me-scan makes efficient use of sequencing output; that pooled libraries are evenly represented in the sequencing results; and that we are able to identify aluyb8/ insertions with a sensitivity and specificity of ~95%  overview of me-scan the key steps of the me-scan protocol are illustrated in figure described below, and detailed in methods. it combines and adapts established methods of transposon display, high-throughput sequencing, and sample indexing. genomic dna is sheared by sonication. the resulting double-stranded dna fragments are repaired to blunt ends, and unpaired adenosines are added to the 3' ends. oligonucleotide adapters with an unpaired 3' thymine are ligated to those fragments. the adapters contain a 5-bp "index" sequence that is unique to each individual sample. this index is later sequenced and links each read pair with the individual from whom it is derived, so samples can be pooled for subsequent steps. at this point, every sequence from every individual's genome is represented by many dsdna fragments. to extract only those fragments that contain an aluyb8/ we perform pcr with a biotinylated primer that anneals to a site found only in aluyb8/ elements and a primer that matches the adapter sequence. this pcr generates some molecules that contain only a fragment of an alu element and no flanking genomic sequence. size selection by gel electrophoresis is used to enrich the resulting pcr product for molecules that contain most of an alu insertion as well as 50- bp of upstream flanking genomic sequence. the biotinylated strands are then bound to streptavidin-coated paramagnetic beads and purified away from nonspecific dna by magnetic separation. the product is re-amplified and checked for quality and quantity. the resulting aluyb8-specific dna fragment library is subjected to paired-end sequencing on one flow cell lane of an illumina genome analyzer ii. instead of using illumina's standard primer for the first sequencing read, we use a custom sequencing primer that anneals to a site bp inside the 5' end of the consensus aluyb8/ sequence. thus the first read will typically consist of bp of aluyb8/ sequence followed by bp of genomic sequence. the second-end read extends from illumina's standard primer and will contain a 5-bp individual-specific index, a't', and bp of genomic sequence. application of me-scan to human aluyb8/ elements yield of usable sequence data *supports one of the insertion loci reported in additional file experimental design we designed two sequencing experiments to test the reproducibility, specificity and sensitivity of me-scan at various levels of sequencing coverage. dna samples from four individuals were used. for the "replication" experiment, we processed two aliquots of dna from individual a in parallel through the ligation step, using a different index for each aliquot, then pooled them in equal amounts for subsequent library preparation and sequencing. for the "pooling" experiment, we prepared five samples with different indexes and combined them in varying proportions to construct a pooled library. figure shows that the proportions of each index as recovered by sequencing match the intended proportions to within 1% on average. thus, it should be possible to pool small amounts of dna from many library preparations without severely under- or over-representing any one library. * only read pairs passing all qc filters are counted. two aliquots of a dna sample from this individual were processed in parallel and pooled for sequencing. a dna sample from this individual was sonicated, then divided into two aliquots for subsequent library construction steps. sensitivity and specificity to estimate the sensitivity of me-scan, we searched for false negative results in our data. we first identified a set of presumably fixed aluyb8/ insertions, reasoning that me-scan should retrieve all of these insertions in every individual, and that failures to do so would likely represent false negative errors. these insertions are present in the human reference genome assembly, contain good matches to the aluyb8/9-specific amplification and sequencing primers we used, are of the canonical aluyb8/ size, and are not known to be polymorphic. we found such loci. the numbers of these loci that were not retrieved in each sample preparation are shown in table on the assumption that each of these loci is indeed fixed for the insertion in humans, the rate of me-scan false negative results ranges from 8% to 25%. the error rate depends on the sequencing effort expended on a sample: the higher the number of reads, the lower the false negative rate. the number of genotypes compared differs from sample to sample due to occasional uncertain genotype calls in the pcr and gel genotyping assays. these genotypes represent actual false negative me-scan results for of the loci, me-scan did not observe the insertion-present allele in any of the samples tested. out of this set of insertion loci, there were loci for which the insertion-present state was not observed by me-scan in any of our four samples. thirty-three of these are part of recently duplicated regions of the genome. read pairs derived from these do not map to a unique position in the genome, and are therefore eliminated by our analysis pipeline. read pairs corresponding to previously unknown alu insertions into such regions would likewise be eliminated and lead to false negative results. another of those insertions lack detectable alu sequence in the bp immediately following the sequencing primer binding site. nearly all of these insertions are in fact observed in the me-scan sequencing results. however, in order to filter out artifactual false positives, the me-scan pipeline discards loci that lack the expected alu sequence, and so these loci lead to false negative results. apart from the loci in recently duplicated regions and those lacking the expected alu sequence in the junction read, it is very likely that some of the putatively fixed aluyb8/ elements are in fact not fixed, and are absent in some of our samples. if so, their absences from the me-scan results do not represent false negatives. we used pcr and gel electrophoresis to genotype loci that were not in duplicated regions and contained the expected alu sequence in the junction read, but still were not retrieved by me-scan in some or all of our samples. the genotypes obtained by pcr and gel-typing for the four individuals are compared with those obtained by me-scan in table in the current me-scan analysis pipeline, alu insertions are treated as presence or absence states for purposes of comparison. of the loci examined, only one had the insertion present in the homozygous state in all four individuals. four more loci had the insertion present in at least some individuals, but the remaining loci were homozygous for the absence of the insertion in all individuals. initially, our sensitivity appears to be ~90% at a sequencing coverage of ~ read pairs per sample pcr genotyping suggests that only ~12% of the putative false negative genotypes that were not in recently duplicated regions are in fact false negatives, so a better estimate of the number of actual false negative results in this experiment is, which implies a sensitivity of %. if we restrict our target set of alu insertions to only those that have intact 5' ends and reside in non-duplicated regions, our sensitivity exceeds 99%. to estimate the specificity of me-scan, we searched for examples of false positive results. any false positives should be in the set of new aluyb8/ insertions retrieved by me-scan, i.e., those that we could not annotate based on the reference genome we identified such insertions. we selected loci at random from this set for confirmation by pcr and gel electrophoresis. all showed the pcr product expected from the alu-filled allele in at least one sample, indicating that the method is very reliable for detecting new insertion loci. in some individuals, however, an insertion detected by me-scan was not detected by pcr and gel electrophoresis, suggesting either that the gel-typing assay was insufficiently sensitive or that there was some cross-contamination between me-scan libraries. on the assumption that the me-scan results are at fault, the false-positive rate ranged from 3% in the highest-coverage samples to 10% in the lowest-coverage samples. the combined biochemical and bioinformatic protocols achieve a specificity of ~96% when based on reads per sample because all individuals were genotyped by me-scan and pcr plus gel electrophoresis for these loci, this data set allows us to examine the rate of false negative me-scan results for this class of loci. no false negatives were observed in the four higher-coverage samples, suggesting that the false negative rate among new alu insertions is as low as that observed for known aluyb8/ loci the data for the three low-coverage samples suggest a higher false-negative rate among new alu insertion loci when relying on lower-coverage sequencing. * not observed in the human reference genome. confirmation of new variable aluyb8/ insertion loci in additional individuals in order to test the dimorphic status of loci in the above-described set of new aluyb8/ insertions, we genotyped them by pcr in a panel of eight additional unrelated individuals: six of east asian descent, one european, and one african individual. the genotyping results are reported in additional file of the loci, showed the expected alu insertion in at least one individual, confirming that they are dimorphic in humans. of the loci that lacked the insertion allele in this panel of eight individuals, one did not show the insertion even in the original panel of four individuals, and three showed the insertion in only one individual of the four. any comprehensive survey of genetic variation will identify "singletons", so our results are consistent with that expectation. these three insertions could be rare in humans, they could be limited to a family or just a single individual, or they might be non-heritable insertions resulting from somatic or even cell-line retrotransposition events. in the full set of new aluyb8/ insertions identified by me-scan, were observed in more than one individual of the original four, which implies that they are polymorphic in humans. the remaining insertions could be rare, de novo, or even non-germline insertions. however, of those loci were among the chosen for further genotyping in the panel of additional individuals, and of those loci, had the insertion present in at least one member of the panel. thus it is likely that a large majority of the new aluyb8/ loci identified by me-scan are in fact polymorphic in humans. reproducibility in order to study the reproducibility of me-scan, we assayed dna from individual a twice, in parallel and with the same level of coverage table shows the results of comparing the alu insertions identified by me-scan in the two replicates. together, the two replicates identified aluyb8/ insertions that are known from the reference genome among these, the proportion of insertions that identified in one replicate but not the other is less than 1%. these replicates also recovered previously known insertions from alu families other than aluyb8/ but with low reproducibility: each replicate missed more than of the insertions identified in the other. the genome contains hundreds of thousands of alu insertions with poor matches to the amplification and sequencing primers we used, so despite the low probability that any one of them will be amplified and sequenced, we expect to see sporadic amplification of some of these loci in each experiment. by the same token, however, the set of loci that are sporadically amplified will differ from sample to sample, so their reproducibility should be low. the two replicates also yielded new alu insertions, with a replication rate of ~92% in that set. this is slightly lower than the ~96% sensitivity observed for the set of known insertions perhaps this set of previously unobserved alu insertion loci includes some from other currently active alu families, such as aluya these loci may amplify erratically due to their poorly matching primer binding sites, thereby reducing the overall reliability in this set. * total positive loci are those observed in either of the two samples of the'replication' experiment, indexed accat and tattc. † number of insertion loci absent in a sample, divided by the total observed in both samples, averaged over the two samples. comparison with previously genotyped aluyb8/ insertion loci numerous researchers, including ourselves, have studied polymorphic alu insertion loci by first ascertaining them in a small set of individuals and then genotyping them in a larger set using locus-specific pcr primers and gel electrophoresis. we previously used this method to genotype polymorphic aluyb8/ loci in the four individuals that we assayed by me-scan in the current work. table shows the results of comparing the genotypes inferred by both methods for those loci in these individuals. most of the disagreements between the two methods are due to negative me-scan genotypes in the low-coverage samples it seems likely that these gel-typing results are correct, and that these disagreements are me-scan false negatives due to insufficient sequencing depth. the remaining four disagreements involve three replicate me-scan genotypes of a single locus in one individual and one genotype at another locus in another individual. me-scan indicates the presence of an insertion in these two cases, while previous genotyping indicated homozygous absent genotypes. the consistency of the me-scan result for the one locus in individual a suggests that the previous genotyping results might be at fault, so we used pcr and gel electrophoresis to check both genotypes we find that, in fact, both genotypes were heterozygous for the presence of the insertion, in agreement with the me-scan results for this set of loci, given sufficient coverage, me-scan appears to be more accurate than gel typing. * all four of the apparent me-scan false positives are due to errors in the previous pcr- and gel-based genotyping. the huref genome sequence of j. craig venter offers the opportunity to verify some of the alu insertions we identified that are absent from the hg19/grch reference. of huref aluyb and aluyb insertions of at least bp in length, were observed by me-scan. although we have no precise expectation for the number of aluyb8/ insertions that our panel of four east asian individuals should share with one european, this result is the equivalent of confirmation by complete sequencing of a sizable set of the previously unobserved loci identified by me-scan. discussion complete ascertainment and genotyping of alu insertion events in large population samples would give us - for the first time - a clear and panoramic view of their population dynamics. knowing the genomic distribution of the most recent alu insertions will allow us to decisively disentangle the effects of target site preferences from the effects of natural selection, recombination, and gene conversion that may subsequently alter that distribution. from the full site frequency spectrum of insertions, we should be able to estimate the transposition rate and the range of selection coefficients that affect alu insertions. it may even be possible to identify factors that cause positive or negative selection on some insertion classes. with sufficient data, we may be able to estimate the evolutionarily relevant, in vivo transpositional activities and effects of specific alu elements by analyzing correlated variation in transposition rates between families and populations. we have developed a method for quickly, sensitively, and specifically ascertaining nearly all aluyb8/ insertions in several individual samples simultaneously. other researchers have used similar targeted high-throughput sequencing approaches to identify me insertions, but with very different goals. typically, an engineered me is used to create many highly uniform insertions of a unique dna construct in a model organism or cell line. selection for a phenotype is then applied, and targeted high-throughput sequencing is used to locate a sample of the selected insertions. me-scan is designed to identify essentially all members of a class of naturally occurring insertions against a large background of very similar sequences in all members of a population sample. by using a fraction of the sequencing capacity of two illumina gaii flow cell lanes to scan four individuals, we have simultaneously identified, mapped, and partially genotyped new variable aluyb8/ insertions in those individuals. nearly all previously known aluyb8/ insertion loci were also detected. we examined several levels of sequencing effort and found that a level of ~ read pairs per sample allowed the assay to achieve ~95% sensitivity and specificity. illumina's genome analyzer iix now yields up to million paired sequence reads per flow cell lane. if this read density is approachable with me-scan libraries, it will be possible to index and pool individuals per lane with very high sensitivity and specificity. even at lower read densities, it should be possible to make more efficient use of the sequencing yield. theoretically, the number of read pairs per insertion should be poisson-distributed, with variance equal to the mean coverage. however, in all our samples, the variances are at least an order of magnitude greater than the means. this is a common feature of high-throughput fragment-library sequencing techniques. reducing this overdispersion even moderately would deliver higher performance at lower levels of sequencing effort by reducing the number of read pairs that are effectively wasted on heavily over-represented insertions. it would also increase the number of reads derived from poorly represented insertions that are at risk of being missed altogether. simple changes to the basic protocol are likely to reduce that overdispersion. the length of sequencing reads has increased along with the number of reads per run. it is now possible to obtain paired bp sequence reads instead of the paired bp reads used here. longer reads would increase both the sensitivity and specificity of the me-scan analysis pipeline. by using a sequencing primer that anneals ~ bp inside the normal 5' end of an alu and reading bp from that primer, one could obtain more of an alu insertion's sequence as well as more immediately adjacent genomic sequence. the additional genomic sequence would allow reliable mapping of some read pairs that are now being discarded. the additional alu sequence would allow the elimination of spurious non-alu products without discarding evidence of alu insertions with modest 5' truncations. a short second read would still be required to sequence the sample index. more importantly, using a long junction read would allow construction of a reference-independent analysis pipeline. the me-scan analysis pipeline described above depends on mapping reads to a reference genome for two reasons. firstly, most of our analyses focused on comparing me-scan results to the "gold standard" information in the reference genome, so mapping was required in any case. secondly, the information in paired bp reads is insufficient for highly accurate reference-independent locus identification and genotyping. the first bp of an alu junction read is usually alu sequence, which - being nearly identical for most alu elements - does not distinguish groups of read pairs that represent different insertion loci. the remaining bp are often insufficient to clearly distinguish all the groups of reads, since some ambiguity must be allowed to handle sequencing errors and some insertions are in regions of low sequence complexity. when mapping read pairs to a reference, those bp combine with the bp of genomic sequence from the flank read to provide enough information for reliable positioning. this allows read pairs to be grouped according to their mapped location. unfortunately, since the bp segments of genomic sequence in the flank reads from a particular insertion are derived from sites at varying distances from the junction read, they will not be identical and may not even overlap. consequently, the flank reads cannot be used to reliably group read pairs based on their sequence alone. however, with a long alu junction read, it should be possible to identify and group all junction reads that represent a particular insertion simply because they will all share nearly identical junction sequences, while reads from different loci will almost always be easily distinguished due to their different sequences it would no longer be necessary to first map every read pair to a reference genome sequence and then group them into loci by their positions. genotyping by grouping read pairs according to the near-identity of their junction sequences should deliver accuracy as good as or better than what we have achieved here. in particular, new insertions into one copy of a duplicated genomic segment will not pose a problem. the position of the new insertion within the duplication will be unique, and all reads corresponding to that insertion will group together, just as they would for new insertions into unique regions. the ability to ascertain and genotype me insertions without relying on a sequenced reference genome would make me-scan very widely applicable: by designing the appropriate amplification and sequencing primers, one could study the population dynamics of nearly any me family in any organism in detail. the method promises to be robust, quick, reliable and relatively inexpensive: the per-bp cost of sequencing continues to decrease; the indexing and pooling method makes efficient use of high-capacity sequencing platforms; library preparation is technically straightforward; the cost of sample preparation is minimized by the use of sonication rather than nebulization, and by pooling many samples for some processing steps; and the amount of template required has decreased by at least fivefold since we began our experiments, further reducing the cost of reagents required for sample preparation. these factors would make a reference-independent me-scan method appealing for simultaneously identifying and genotyping polymorphic markers of population genetic diversity in any species.  me-scan enables the rapid, efficient, and highly accurate ascertainment and genotyping of thousands of mobile element insertion loci of a particular subfamily in many individuals, even in the presence of a very large background of related elements. with longer sequencing read lengths, it will be possible to apply me-scan to species for which no full-genome reference sequence is available. the ability to quickly and inexpensively gather so much detailed information on any me family in any population represents a breakthrough in the study of the population dynamics and evolution of mobile elements. 
here we present a novel method for identifying nearly all insertions of a me subfamily in the whole genomes of multiple individuals and simultaneously genotyping those insertions that are variable in the population. mobile element insertion loci of the target subfamily are uniquely identified by their junction sequence, and all insertion junctions are linked to their individual libraries by the corresponding index sequence. research on the impacts, population dynamics, and evolution of mes is constrained by the difficulty of ascertaining rare polymorphic me insertions that occur against a large background of pre-existing fixed elements and then genotyping them in many individuals. in light of recent improvements to high-throughput sequencing technology, it should be possible to employ me-scan to genotype insertions of almost any mobile element family in many individuals from any species. mobile elements are diverse, common and dynamic inhabitants of nearly all genomes. mobile element scanning is an efficient method for quickly genotyping mobile element insertions with very high sensitivity and specificity. in four individuals, we identified a total of aluyb and aluyb insertions, including nearly all those that are present in the reference genome, as well as that are not. individual-specific "index" sequences are designed into the oligonucleotide adapters used to construct the individual libraries.
 mobile elements are diverse, common and dynamic inhabitants of nearly all genomes. me transposition generates a steady stream of polymorphic genetic markers, deleterious and adaptive mutations, and substrates for further genomic rearrangements. research on the impacts, population dynamics, and evolution of mes is constrained by the difficulty of ascertaining rare polymorphic me insertions that occur against a large background of pre-existing fixed elements and then genotyping them in many individuals.  here we present a novel method for identifying nearly all insertions of a me subfamily in the whole genomes of multiple individuals and simultaneously genotyping those insertions that are variable in the population. we use me-specific primers to construct dna libraries that contain the junctions of all me insertions of the subfamily, with their flanking genomic sequences, from many individuals. individual-specific "index" sequences are designed into the oligonucleotide adapters used to construct the individual libraries. these libraries are then pooled and sequenced using a me-specific sequencing primer. mobile element insertion loci of the target subfamily are uniquely identified by their junction sequence, and all insertion junctions are linked to their individual libraries by the corresponding index sequence. to test this method's feasibility, we apply it to the human aluyb and aluyb subfamilies. in four individuals, we identified a total of aluyb and aluyb insertions, including nearly all those that are present in the reference genome, as well as that are not. index counts show the sequenced products from each sample reflect the intended proportions to within 1%. at a sequencing depth of paired reads per sample, the sensitivity and specificity of me-scan are both approximately 95%.  mobile element scanning is an efficient method for quickly genotyping mobile element insertions with very high sensitivity and specificity. in light of recent improvements to high-throughput sequencing technology, it should be possible to employ me-scan to genotype insertions of almost any mobile element family in many individuals from any species. 