BACKGROUND
the completion of the human and of non-human primate reference genome sequences showed that nearly half of the genome is derived from various transposable sequences .
due to their ability to move within the genome active retrotransposons represent an important source of genomic structural polymorphisms .
retrotransposition involves rna intermediates inserting via the target-primed reverse transcription mechanism   <cit> .
the tprt process produces short  target site duplications  at the flanks of the newly integrated elements  <cit> .
most mobile element activity in humans results from non-ltr retrotransposons including alu, l <dig> and sva  <cit> .
upon transcription, the rna polymerase may skip weak transcription termination signals  signal, 5′-aataaa-3′ for l <dig> and sva), and hence terminate rna synthesis at a polya signal further downstream   <cit> .
this process can lead to 3′ transductions at l <dig> and sva elements, causing mobilization of downstream flanking sequence together with the mobile element .
in addition, short sequence transductions  can occur when the rna cleavage-prior-to-polyadenylation occurs abnormally and slightly downstream than usual, capturing a small piece of the genomic location after the short poly-a track of the source l <dig> element  <cit> .
retrotransposition can contribute to diseases  <cit>  and evolution  <cit>  and previous studies identified differences in the spectrum of mobile element classes among distinct primate species  <cit> .
transductions play an important role in this process, e.g.
through mobilization of genomic functional elements including exons or by resulting in gene disruption events .
previous studies focusing on reference transductions  have reported that transductions are relatively abundant, with estimates that around 10 % of l <dig> and sva insertions detectable in the human reference assembly exhibit 3′ transduction events .
only few recent studies, however, have investigated non-reference transductions and consequently there is little knowledge on transduction-mediated sequences polymorphic in the population.
kidd and co-authors, prior to the widespread application of next generation sequencing , identified several polymorphic l1-transductions through a fosmid library-based sanger sequencing approach in nine hapmap samples  <cit>  and macfarlane and co-authors developed the experimental ts-atlas method that uses l <dig> 3′ transductions as sequence tags to identify active l <dig> lineages in a genome-wide context  <cit> .
furthermore, more recently, tubio and colleagues reported an abundance of somatic l <dig> transduction events in cancer genomes sequenced with short dna reads  <cit> , paterson and colleagues identified 3′ transduced sequences in oesophageal adenocarcinomas  <cit>  and two studies recently reported somatic l <dig> insertions with 5′ and 3′ transductions in human neurons  <cit>  – which highlights that somatic transductions can occur outside of cancer and may be relevant for a broader range of diseases.
detecting variants in somatic genomes, however, is conceptually different from germline polymorphism inference, and polymorphic transduction events arising in germline genomes have – to the best of our knowledge – not systematically been studied by ngs thus far.
here we describe a computational approach suitable for the discovery of non-reference polymorphic  mobile element transduction events – termed tiger for transduction inference in germline genomes – based on illumina ngs data.
we applied tiger to the detection of l <dig> mediated 3′-transductions, the most abundant class of mobile element transductions  <cit> , in five chimpanzee, five orangutan and five macaque  <cit>  samples sequenced to a mean coverage of ~20x as well as to the well-characterized human na <dig> lymphoblastoid cell line  <cit> .
furthermore, we performed extensive experimental validation and event characterization by pcr and state of the art single-molecule long dna read sequencing technologies.
our analyses demonstrate differences in the rate of transduction across primate species, and highlight species-specific mobile element subfamilies involved in l <dig> transduction.
tiger, made available open source , makes a relevant class of structural variation amendable for personal genome analysis.
methods
whole-genome sequencing data
using tiger we analyzed previously published chimpanzee, orangutan and macaque whole-genome sequencing  data  <cit>  from five individuals per species, sequenced between  <dig> - <dig> x, as well as the human na <dig> sample down-sampled to ~20x   <cit> .
details on read mapping and filtering are in the supplementary methods .
tiger specifications
tiger uses a combination of  non-reference l <dig> insertions – in this study discovered by a modified version of tea  <cit> , including lower-confidence l <dig> elements inferred by tea, to allow for increased sensitivity   <cit> ,  translocation  calls identified using the delly  <cit>  translocation detector module as well as  single-anchored  reads obtained directly from bam  files.
sa and tl reads are found as discordantly mapped read pairs, either having one read unmapped or placed randomly due to the mapping ambiguity , or both reads in a pair mapped onto two different chromosomes   <cit> .
overlap between non-reference l <dig> insertion and tl reads is used as evidence by tiger to infer the presence of l1-mediated transductions.
the search space of each insertion locus was increased by 500 bp on either side  to define the candidate region.
each discordant  read mapping onto l1-mediated transduction candidate regions was obtained and respective mates realigned onto the corresponding reference genome to identify possible element sources .
this additional realignment step was carried out using blat  <cit>  .
at least 50 bp of each realigned tl or sa mate  was required to ensure robust mapping to the reference genome.
furthermore, realigned mates were processed based on the highest bit-score and the total number of possible matches  to find the best reference match and to differentiate repetitive regions  from unique regions , respectively.
we required clustering of at least four dna sequence reads  on the same source chromosome, which enabled us to construct extended sequence stretches that better reflect the portion of unique dna sequence transduced, whereas clusters of repetitive reads mapping randomly multiple times in the genome were used to infer l <dig> presence.
in line with the sequencing coverage of our data, as well as our observations from manual inspections and experimental validations, the upper limit of clustered reads at one source locus was calibrated to the value of  <dig>  in order to bypass regions containing solo repetitive mobile elements and regions exhibiting a remarkably high number of supporting reads .
to ensure that predicted transduction sequences are unique, the mean of all read-specific tm values per locus was set to be ≤ <dig>  once all aforementioned steps were satisfied, the longest possible stretch of each unique source locus was extended by utilizing reads clustering in an overlapping fashion, and without gaps and reported as the computationally predicted transduced sequence.
all predicted l1-mediated transduction insertion regions were filtered for overlap with corresponding segmental duplications  as well as the presence of a reference l <dig> at the insertion to avoid false positives .
tsds were extracted directly from the annotation of previously detected l <dig> elements , whereas the putative presence of a polyadenylation tail  was evaluated by searching for six consecutive non-reference a′s or t′s  in each read.fig.
<dig> tiger approach.
a l1-mediated transduction insertions are typically composed of flanking target site duplications , l <dig> sequence and unique transduction sequence  followed by a non-reference polya tail.
to detect such events in paired-end ngs data, candidate regions are chosen based on an overlap between l <dig> insertion loci, paired-ends indicative for an insertion of unique sequence copied from a distal locus  supporting read pairs), and remapped single-anchored  reads in the reference genome.
b a combination of reads indicative for l <dig> insertion as well as unique duplicative sequence insertion and additionally single-anchored reads are used to discover l1-mediated transduction insertions.
tl and sa read pairs are realigned to ensure correct placement onto the reference genome.
additional filtering steps are implemented for removal of low-confidence calls
l <dig> subfamily assessment
to assess which subfamily class drives l <dig> insertions as well as l1-mediated transductions, consensus sequences for all full-length  primate l1s were constructed from multiple reference elements .
to discover l <dig> subfamilies driving the transduction, longer contig sequences assembled from short reads were realigned to the primate-specific l <dig> consensus sequences.
a best mapping criterion was used to infer the most probable l <dig> subfamily involved in transduction at each locus.
pcr and minion based experimental validations
experimental validations of l1-mediated transduction predicted loci were performed using pcr coupled with capillary sequencing, as well as by single molecule long dna read sequencing .
human tiger calls were validated using a pacbio whole genome sequencing dataset of na <dig>  <cit> .
oxford nanopore minion data was generated based on long-range pcr amplicons, using a sequencing platform that we acquired as part of the minion early-access programme.
RESULTS
computational discovery of l1-mediated transductions through tiger
tiger scans genomic regions for the presence of three mobile element transduction-defining signatures:  characteristic hallmarks of mobile element insertions , including non-reference polya tails and tsds, which are retrieved from the transposable element analyzer  algorithm  <cit> ;  aberrant mapping of paired-ends indicative of the adjacent  insertion of an additional unique sequence originating from a distal locus ;  single-anchored paired-end reads  that are reassessed by tiger to further substantiate insertion signals .
the modular nature of tiger enables it to be applied with any tool for polymorphic mobile element insertion detection.
subsequently, tiger pursues additional event characterization steps including realignment, read clustering and filtering to identify high confidence transduction events .
to test tiger’s utility for detecting polymorphic l1-mediated 3′ transductions, we applied the tool to fifteen recently published genomes from three non-human primate species   <cit> .
an example transduction event detected by tiger is depicted in fig.
<dig>  this event involves an inter-chromosomal duplicative insertion of a unique sequence of chimpanzee chromosome  <dig>  into the respective target region  mediated by an l1-driven transduction.
by realignment onto the reference genome with blat  <cit>  we placed previously unmapped  reads onto the reference genome facilitating characterization of the l1-mediated transduction, as visualized in fig.
2a.
a more detailed view of read placements is provided in fig.
2b, with one read mapping to the target locus on chromosome  <dig> and the other read  either mapping to a non-reference l <dig> element  or forming a cluster of reads uniquely mapping to the source on chromosome  <dig> .
some of these latter read mates contain the non-reference polya tail and target site duplication .
the additional polya signal , causing termination of the transduced sequence during the transcription process, is also visible in the data .
we additionally evaluated the sensitivity of tiger for predicting 3′ l1-mediated germline transductions in ngs data by performed in silico simulations , estimating a sensitivity of 86 %.fig.
<dig> computational analysis of the chr7:6620368- <dig> insertion into the chr10:54643580- <dig> region in the chimpanzee sample pr <dig>  a depiction of the chr10:54643580- <dig> region using the integrative genomics viewer   <cit>  before read realignment .
after realignment using blat many initially single-anchored reads were placed correctly, facilitating the ascertainment of this l1-mediated transduction clustering to a region on the source chromosome  <dig> with an average uniqueness of  <dig> .
b a detailed view of l1-mediated 3′ transduction read placements: one read is shown to map to the target locus on chromosome  <dig> and the other read  maps either to a non-reference l <dig> element  or forms a cluster of reads uniquely mapping to the source on chromosome  <dig> .
out of  <dig> reads,  <dig> were carrying parts of a non-reference polya tail
experimental verification of transductions by pcr and capillary sequencing
to verify the accuracy of tiger, we performed validation experiments on  <dig> randomly chosen 3′ transduction calls , using pcr followed by capillary sequencing .
we employed a combination of an outer and inner primer pair to specifically amplify the target region, and to overcome the barriers brought about by the two respective polya tails for pursuing validation by capillary sequencing .
this validation strategy enabled verification of both the presence of the mei and of the transduced unique sequence.
a representative pcr gel picture for macaque, using outer primers, is shown in fig.
3b.
a circos plot depicting all predicted transductions in macaque  is provided in fig.
3c.
in total, we verified  <dig> out of 51 l1-mediated transduction calls, based on which we estimated a false discovery rate   of  <dig>  % .
investigation of the experimental data on the eight false positive loci revealed that seven lacked meis  as well as the transduced sequence, whereas the remaining locus presented evidence of an l <dig> insertion but lacked the inferred transduced sequence .table  <dig> summary of tiger results in non-human primate species
for comparison to na <dig> see additional file 1: table s5
*determined based on ratio between tiger transductions and l <dig> insertions.
95 % confidence intervals were calculated using one sample t-test
**significantly different based on wald test of predicted-transduction rates: chimpanzee-macaque: p =  <dig> ; chimpanzee-orangutan: p =  <dig> ; macaque-orangutan: p =  <dig>
fig.
<dig> experimental verification of tiger-based l1-mediated 3′ transductions by pcr.
a general primer design: outer  primers were placed outside of the event in the target locus to amplify the l1-mediated sequence transduction insertion allele and/or the reference genome allele.
on the left side of the locus, the corresponding sequence  uniquely matches the target site, and subsequently matches to multiple positions in the genome in line with the presence of an l <dig> element.
further to the right, the sequence will also match uniquely to the target site and end with a polya stretch not seen in the reference genome.
in order to confirm the presence and origin of the transduced sequence , we employed a 2nd set of primers  inside the predicted unique transduction sequence.
b example pcrs verifying rhesus macaque l1-mediated sequence transductions, based on outer primers, are shown for inferred carrier  and non-carrier  samples.
in the presence of an l1-mediated transduction sequence insertion, a larger band than the reference band in nc is seen; heterozygotes show both bands whereas homozygous l1-mediated sequence transduction insertions show only one  band.
c a circos plot shows the distribution for all inferred rhesus macaque l1-mediated sequence transductions ; experimentally validated insertions by pcr and minion single molecule sequencing are depicted in green.
arrowheads indicate directionality towards the target locus
tiger can be used for estimation of the size of the transduced sequence – however, this capability is strongly dependent on read length and insert-size of the paired-end ngs library, with short ngs reads being only of limited use for detecting the boundaries of the l <dig> element’s 3′ and the transduced sequence’s 5′ at target loci.
in our analyses, sizes of computationally inferred transduction sequence lengths varied between 90–260 bp in chimpanzee, 74–437 bp in orangutan and 64–361 bp in macaque.
this suggests that tiger’s minimal sequence requirement for reliably inferring transductions in illumina sequencing data is ~50–60 bp.
verification and characterization of transductions by single molecule sequencing
both short ngs reads and sanger sequencing reads do not typically fully span the target locus, which complicates the characterization of transduction events.
we reasoned that third generation long-read single molecule dna sequencing technologies may help overcome this challenge, by fully recovering the complete sequence and structure of the combined insertion.
hence, we employed both pacific biosciences  sequencing  <cit>   and oxford nanopore minion sequencing  to obtain further insights into l1-mediated transductions.fig.
<dig> pacific biosciences  and oxford nanopore minion  long read verification of l1-mediated transduction insertions.
a
left panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671- <dig> region shown on the x-axis; pacbio read on the y-axis: ~1000 bp shift shows presence of insertion.
right panel: inspection of the inserted sequence verified the presence of the l <dig> element  and the transduced sequence including the new polya tail ; the new polyadenylation signal is underlined.
b dotplot – with reference genome sequence on the x-axis and minion read on the y-axis: ~1200 bp shift shows presence of an insertion.
the inserted sequence verified both the presence of an l <dig> element  and additional transduced sequence including the new polya tail .
c alignment of the inserted l <dig> sequence to the ~6 kb long l <dig> consensus sequence shows that the integrated l <dig> is 5′-truncated
we first employed tiger to discover transduction events in the human hapmap dna sample na <dig> , which enabled us to overlay tiger transduction calls with long dna sequencing read data  previously generated by whole genome sequencing  using pacbio technology  <cit> .
our analysis of the data showed that long dna reads are indeed valuable for characterization of l <dig> transductions.
as an example, alignment of a 7 kb long pacbio read from na <dig> to a transduction candidate locus on chromosome  <dig>  demonstrated a ~1 kb shift in the alignment, further substantiating the presence of the insertion predicted by tiger .
additional inspection of the pacbio sequence allowed us to characterize the structure of the event in more detail .
indeed, analysis of the inserted sequence verified the presence of an l <dig> 3′ transduction, with a 908 bp 5′-truncated l <dig> element exhibiting a 126 bp long transduced sequence ending with a polya tail in 3′.
using the previously published pacbio wgs data for na <dig>  <cit>  as well as fosmid sequencing data generated previously for na <dig>  <cit> , we verified four out of six l1-mediated transductions identified in this human sample .
from the remaining two human events, one showed a solo l <dig> insertion  upon further inspection of the pacbio reads, whereas the other locus remained inconclusive, as it lacked coverage of pacbio reads at the genomic region in question, preventing us from verifying the element by computational means.
second, we obtained similar validation results for all three non-human primate species, through oxford nanopore sequencing data which we generated as part of the minion early-access programme, following long-range pcr amplification of candidate loci .
for example, minion reads spanning the rhesus macaque l <dig> transduction candidate locus on chromosome  <dig>  verified the presence of a ~ <dig>  kb insertion , and further analysis of the inserted sequence demonstrated a 116 bp long 5′-truncated l <dig> element and transduction of 1043 bp of additional sequence including the new polya tail in 3′ .
overall, we validated  <dig> transductions by single molecule sequencing , which combined with the  <dig> pcr validations  resulted in  <dig> experimentally validated l <dig> transductions – to our knowledge the largest dataset on validated non-reference germline mobile element transductions reported to date .
facilitated by the generated long read sequencing data we examined the length distribution of the inserted l <dig> elements and of the transduced sequences, observing transduction sequence lengths ranging from  <dig> to 1570 bp .
our validation experiments further verified an abundance of 5′ l <dig> sequence truncations as previously reported in a similar context  <cit> .
among  <dig> experimentally validated transductions, most showed relatively small l <dig> elements .
investigation of transduction rates in primate species
we further made use of inferred transduction events to estimate rates of transduction in different non-human primate species, encouraged by earlier studies demonstrating differential activities of solo mobile element insertions across primate species  <cit> .
tiger altogether detected  <dig>  non-redundant l1-mediated 3′ transductions in the  <dig> primate genomes,  <dig> in rhesus macaque,  <dig> in orangutan and  <dig> in chimpanzee .
average numbers of l1-mediated transductions per individual were  <dig>  for macaque,  <dig>  in orangutan and  <dig>  in chimpanzee.
to calculate the rate of transductions per species, we divided the number of high confidence tiger transduction calls by the total number of non-reference l <dig> insertions  identified using tea  <cit>  or tiger .
our transduction rate estimates were significantly different between species with estimates of  <dig>  % ±  <dig>  ci  for chimpanzee,  <dig>  % ±  <dig>  for orangutan, and  <dig>  % ±  <dig>  for macaque, , chimpanzee-macaque  and orangutan-macaque , table 1).
we further tested whether the observed difference in transduction rates among species could reflect underlying differences in the efficacy of selection among non-human primates with different effective population sizes.
we observed no evidence that selective constraints varied substantially among these primate species, and obtained little evidence for an impact of effective population size on the efficacy of selection .
the total amount of l <dig> calls in the human na <dig> down-sampled genome was  <dig>  – resulting in a transduction rate estimate of ~ <dig>  % .
similarly, filtering the kidd et al.
data  <cit>  for calls overlapping with l <dig> elements in the target regions  and requiring more than 50 bp of uniquely mappable transduced sequence, resulted in an adjusted transduction rate estimate of  <dig>  % .
lastly, we examined whether tiger detected 5′ transductions in our primate dataset.
notably, we did not observe evidence for a single l <dig> 5′ transduction event driven by a new upstream promoter, which is consistent with earlier reports based on reference transduction analysis suggesting a very low rate of such events  <cit> .
characterization and l <dig> subfamily analysis of transduction events
to investigate potential functional consequences of transductions we analysed the overlap of transduced sequences  with annotated functional regions of the genome, considering all events identified in this study.
the majority of transduction sequences were derived from intergenic regions  and a similar fraction also inserted into intergenic regions.
approximately a third  inserted into intronic regions, where some may have an effect on gene regulation  <cit>  .
intersection of source coordinates of inferred transductions with exonic annotations, furthermore, identified two candidate events – one in orangutan and one in macaque.
in both cases, while there was strong evidence for the insertion of unique genomic sequence, evidence for l <dig> associated sequence signatures was minimal.
notably, following further manual inspection and pcr validation, both insertions turned out to represent gene retrocopy insertion polymorphisms   <cit>  rather than transduction events.
grips share many diagnostic features with transductions, such as a tsd and the insertion of unique sequences, including the presence of a polya tract, as they are mobilized by the l <dig> machinery in trans  <cit> , which may explain why tiger was able to identify these events in this context .
we further investigated the source-donor l <dig> relationship with a focus on transduced sequences.
among three out of the six transduction calls observed in humans, we obtained evidence for a full-length reference l <dig>  element immediately upstream of the predicted transduction source locus .
contrary to the human genome, we saw no clear indication confirming the presence of full length donor l <dig> elements at source loci within non-human primates, apart from one source region in rhesus macaque exhibiting a >5 kb long l1pa <dig> element within a 10 kb region surrounding the transduced source sequence.
we therefore classified the source loci of all validated transductions into two classes:  no donor l <dig> fragments annotated within 5 kb to either side of the transduced sequence  and  presence of small, truncated l <dig> elements surrounding the transduced sequence, either unrelated to the transduction or severely truncated following the formation of the transduction event .
our analysis suggests that the majority of calls belong to class , i.e.
<dig> in rhesus macaque,  <dig> in orangutan and five in chimpanzee, whereas the remainder fall into class  .
in addition, our analysis did not reveal any hotspot donor l1s generating multiple transductions, as recently described in an in-depth analysis of somatic transduction events in cancer genomes  <cit> .
finally, we investigated l <dig> subfamilies responsible for transductions to evaluate whether l <dig> subfamily specificity may explain the differential rate at which l <dig> elements are accompanied by transduced sequences in different species .
to this end we remapped contigs assembled from short reads aligning to the inserted l <dig> sequence to the consensus l <dig> subfamilies and enumerated best alignment hits .
notably, 96 % of all annotated l1-mediated transduction insertions in rhesus macaque belonged to the l1cer family , which has evolved from macaque-specific l1pa <dig>  <cit> .
in orangutan, by comparison, 63 % of the annotated l1-mediated sequence transductions were associated with the l1pa <dig> subfamily and 92 % with any l1pa family member.
furthermore, in chimpanzee all transductions were found in association with l1pt members.
interestingly, we observed examples where l1s accompanied by transductions showed a different subfamily distribution than solo l1s in the respective species .
in macaque, for example, l1cer- <dig> showed a slight enrichment for transductions , albeit not nominally significant, whereby l1cer- <dig> associated transductions showed depletion relative to solo l1s .
by comparison, in orangutan l1pa <dig> elements exhibited an increased rate of transductions relative to solo l1s , whereby in chimpanzee too few l <dig> transductions with reliable subfamily annotation were identified to allow for robust statistics.fig.
<dig> l <dig> subfamilies associated with l1-mediated transductions: p values are based on fisher’s exact test per subfamily using 2 ×  <dig> contingency tables
discussion
retrotransposon mediated transductions are an important class of polymorphic structural variation in the germline, which so far has been largely unexplored, and tiger renders this class of genetic variation amenable to ngs-based analyses.
while the detection of transductions presents technical challenges in short read dna sequencing data, owing to the repetitive nature of mobile elements  <cit>  and the fact that illumina sequencing reads are short when compared with l <dig> sequences, we have demonstrated tiger’s ability to robustly identify l1-transduced sequence elements.
our data indicate variability in transduction rates between species, with rhesus macaque and chimpanzee exhibiting significantly reduced transduction rates  compared to orangutan , which adds to previous findings of differential activities of mobile elements among primate species  <cit> .
we note that a number of l <dig> insertions associated with transductions in the samples covered by our study were previously overlooked in a scan for solo l <dig> insertions  <cit> , presumably since in the presence of a transduction these events lacked sufficient evidence from both 5′ and 3′ flanks for solo l <dig> inference – which indicates that application of tiger can increase sensitivity for l <dig> detection.
differences in transduction rate may have evolutionary consequences, given that transductions can mobilize functional genomic dna sequences  <cit> .
these differences may at least in part be mediated by l <dig> subfamily usage.
our transduction rate estimate for humans  is slightly lower when compared to previous studies reporting ~10 %  <cit> , a difference that may be attributed to the stringent filtering we performed.
kidd et al.
<cit>  identified transductions accompanying 20 % of all non-reference l1s, resulting in a higher transduction rate – but when we employed equivalent filters used in conjunction with tiger on the kidd et al.
data, we obtained a comparable transduction rate estimate of  <dig>  %.
it should be stressed that we designed tiger for the analysis of short  ngs reads, which are known to map ambiguously in the context of repetitive genomic sequence when compared to pacbio or capillary sequencing reads – and remaining limitations related to the use of illumina data exist across methods utilizing short reads.
thus tiger may be insensitive to elements that insert into or derive from regions of low mappability  <cit> , as well as to transductions <50 bp in length.
another limitation of our study is that we did not specifically investigate orphan transductions arising from the same process  <cit> .
it is possible that an improved fdr of tiger may be achieved in conjunction with higher confidence mei calls.
in this regard, our fdr estimate for tiger  is well in line with a recent fdr estimate  for the tea mobile element discover algorithm  <cit>  used for inferring mei signals in our study  <cit> .
furthermore, our investigation of pacbio and minion single molecule dna long read sequencing data demonstrates the potential of third generation sequencing for uncovering such events, with the promise to facilitate identification of these also in more repetitive regions of the genome as already suggested in a study by chaisson et al.
<cit> , once such technologies are more widely applied at a genome-wide scale.
although tiger should in principle be capable of identifying 3′ and 5′ transductions accompanying l <dig> insertions, we have not observed a single 5′ transduction event driven by an alternative upstream promoter in our data.
5′ transduction events were shown to be extremely rare in human genomes with only few cases reported  <cit> .
scarcity of 5′ l <dig> transductions may also relate to the common truncation of the 5′ part of the transcript  observed during the tprt based integration mechanism, which would be expected to particularly affect 5′ transduction sequences upstream of the l <dig> element.
a recent study of somatic l <dig> transduction events in cancer genomes is consistent with a scarcity of 5′ l <dig> transductions  <cit> .
cancer-associated transductions, furthermore, have been reported to be typically highly clustered, whereby a single source l1-master element tends to cause several transduction events in unrelated samples  <cit> .
interestingly, we did not observe such clustering in the samples studied here, perhaps due to relaxed suppression of active l <dig> elements in the germline, which may reduce event clustering.
in somatic tissues most of active l <dig> sources are suppressed, and clustering may occur when one or few escape from the suppression, mediated, for example, by local alterations in chromatin structure and dna methylation in cancer cells.
our study focused on the inference of polymorphic l <dig> transductions, with l <dig> elements belonging to the autonomous retrotransposition-competent mobile element class commonly mobilizing non-repetitive sequences.
while sva elements are also capable of transducing unique dna sequences  <cit>  and therefore could be a future scope of further tiger development, they are still absent from the macaque genome  <cit> , and previously observed novel non-reference sva elements in other species were relatively small in number  <cit> .
alu-mediated transductions have so far not been reported in the literature and thus were not a target of our study.
in principle, while we chose to work with the tea algorithm to identify l <dig> input signals, tiger can be used in conjunction with other mei discovery algorithms, including tangram  <cit> , mobster  <cit> , te-tracker  <cit>  and retroseq  <cit> , augmenting the functionality of these existing tools.
by mobilizing unique sequences, l <dig> elements can shuffle and duplicate potentially functional genomic segments adding to genomic diversity.
tiger enables investigation of this relevant layer of genetic diversity in personal genomes, with potential future applications to disease and evolutionary studies.
CONCLUSIONS
we developed tiger for identifying non-reference retrotransposon-mediated transductions in the germline using ngs data.
tiger, which can be used in conjunction with a number of translocation and non-reference retrotransposon discovery tools, will enhance variant analysis pipelines, and offers access to an as yet under-explored type of germline genetic variation.
ethics and consent to participate
not applicable.
consent to publish
not applicable.
availability of data and materials
all data presented in this study is either a part of the manuscript or supplementary files listed below.
additional files
additional file 1: contains all supplemental material and methods and supplemental figures and tables.
additional file 2: is a list of all l1-transductions discovered in this study and their validation status in macaque, orangutan, chimpanzee and human.
additional file 3: contains a list of all l1-transductions discovered in this study near genes that either originate from an intronic sequence or insert into an intronic sequence in macaque, orangutan, chimpanzee and human.
additional file 4: contains igv representations from each experimentally validated source locus .
abbreviations
fdrfalse discovery rate
gripgene retrocopy insertion polymorphism
memobile element
meimobile element insertion
ngsnext generation sequencing
pa/polyapolyadenylation
sasingle-anchored
svstructural variant
tltranslocation
tstransduced sequence
tsdtarget-site duplication
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jt, ams and jok designed the study.
jt developed tiger.
el and pjp performed l <dig> detection and subfamily analyses.
og, au and ams performed validation experiments or validation experiment analysis.
sm designed and executed in silico transduction simulations.
dg and eemf provided population genetics analyses.
jt, el, ams and jok made contributions to writing the manuscript, with additional input from all remaining authors.
all authors read and approved the final manuscript.

 the completion of the human and of non-human primate reference genome sequences showed that nearly half of the genome is derived from various transposable sequences. due to their ability to move within the genome active retrotransposons represent an important source of genomic structural polymorphisms. retrotransposition involves rna intermediates inserting via the target-primed reverse transcription mechanism. the tprt process produces short target site duplications at the flanks of the newly integrated elements. most mobile element activity in humans results from non-ltr retrotransposons including alu, l and sva. upon transcription, the rna polymerase may skip weak transcription termination signals signal, 5′-aataaa-3′ for l and sva), and hence terminate rna synthesis at a polya signal further downstream. this process can lead to 3′ transductions at l and sva elements, causing mobilization of downstream flanking sequence together with the mobile element. in addition, short sequence transductions can occur when the rna cleavage-prior-to-polyadenylation occurs abnormally and slightly downstream than usual, capturing a small piece of the genomic location after the short poly-a track of the source l element. retrotransposition can contribute to diseases and evolution and previous studies identified differences in the spectrum of mobile element classes among distinct primate species. transductions play an important role in this process, e.g. through mobilization of genomic functional elements including exons or by resulting in gene disruption events. previous studies focusing on reference transductions have reported that transductions are relatively abundant, with estimates that around 10 % of l and sva insertions detectable in the human reference assembly exhibit 3′ transduction events. only few recent studies, however, have investigated non-reference transductions and consequently there is little knowledge on transduction-mediated sequences polymorphic in the population. kidd and co-authors, prior to the widespread application of next generation sequencing, identified several polymorphic l1-transductions through a fosmid library-based sanger sequencing approach in nine hapmap samples and macfarlane and co-authors developed the experimental ts-atlas method that uses l 3′ transductions as sequence tags to identify active l lineages in a genome-wide context. furthermore, more recently, tubio and colleagues reported an abundance of somatic l transduction events in cancer genomes sequenced with short dna reads, paterson and colleagues identified 3′ transduced sequences in oesophageal adenocarcinomas and two studies recently reported somatic l insertions with 5′ and 3′ transductions in human neurons – which highlights that somatic transductions can occur outside of cancer and may be relevant for a broader range of diseases. detecting variants in somatic genomes, however, is conceptually different from germline polymorphism inference, and polymorphic transduction events arising in germline genomes have – to the best of our knowledge – not systematically been studied by ngs thus far. here we describe a computational approach suitable for the discovery of non-reference polymorphic mobile element transduction events – termed tiger for transduction inference in germline genomes – based on illumina ngs data. we applied tiger to the detection of l mediated 3′-transductions, the most abundant class of mobile element transductions, in five chimpanzee, five orangutan and five macaque samples sequenced to a mean coverage of ~20x as well as to the well-characterized human na lymphoblastoid cell line. furthermore, we performed extensive experimental validation and event characterization by pcr and state of the art single-molecule long dna read sequencing technologies. our analyses demonstrate differences in the rate of transduction across primate species, and highlight species-specific mobile element subfamilies involved in l transduction. tiger, made available open source, makes a relevant class of structural variation amendable for personal genome analysis. methods whole-genome sequencing data using tiger we analyzed previously published chimpanzee, orangutan and macaque whole-genome sequencing data from five individuals per species, sequenced between - x, as well as the human na sample down-sampled to ~20x. details on read mapping and filtering are in the supplementary methods. tiger specifications tiger uses a combination of non-reference l insertions – in this study discovered by a modified version of tea, including lower-confidence l elements inferred by tea, to allow for increased sensitivity, translocation calls identified using the delly translocation detector module as well as single-anchored reads obtained directly from bam files. sa and tl reads are found as discordantly mapped read pairs, either having one read unmapped or placed randomly due to the mapping ambiguity, or both reads in a pair mapped onto two different chromosomes. overlap between non-reference l insertion and tl reads is used as evidence by tiger to infer the presence of l1-mediated transductions. the search space of each insertion locus was increased by 500 bp on either side to define the candidate region. each discordant read mapping onto l1-mediated transduction candidate regions was obtained and respective mates realigned onto the corresponding reference genome to identify possible element sources. this additional realignment step was carried out using blat. at least 50 bp of each realigned tl or sa mate was required to ensure robust mapping to the reference genome. furthermore, realigned mates were processed based on the highest bit-score and the total number of possible matches to find the best reference match and to differentiate repetitive regions from unique regions, respectively. we required clustering of at least four dna sequence reads on the same source chromosome, which enabled us to construct extended sequence stretches that better reflect the portion of unique dna sequence transduced, whereas clusters of repetitive reads mapping randomly multiple times in the genome were used to infer l presence. in line with the sequencing coverage of our data, as well as our observations from manual inspections and experimental validations, the upper limit of clustered reads at one source locus was calibrated to the value of in order to bypass regions containing solo repetitive mobile elements and regions exhibiting a remarkably high number of supporting reads. to ensure that predicted transduction sequences are unique, the mean of all read-specific tm values per locus was set to be ≤ once all aforementioned steps were satisfied, the longest possible stretch of each unique source locus was extended by utilizing reads clustering in an overlapping fashion, and without gaps and reported as the computationally predicted transduced sequence. all predicted l1-mediated transduction insertion regions were filtered for overlap with corresponding segmental duplications as well as the presence of a reference l at the insertion to avoid false positives. tsds were extracted directly from the annotation of previously detected l elements, whereas the putative presence of a polyadenylation tail was evaluated by searching for six consecutive non-reference a′s or t′s in each read.fig. tiger approach. a l1-mediated transduction insertions are typically composed of flanking target site duplications, l sequence and unique transduction sequence followed by a non-reference polya tail. to detect such events in paired-end ngs data, candidate regions are chosen based on an overlap between l insertion loci, paired-ends indicative for an insertion of unique sequence copied from a distal locus supporting read pairs), and remapped single-anchored reads in the reference genome. b a combination of reads indicative for l insertion as well as unique duplicative sequence insertion and additionally single-anchored reads are used to discover l1-mediated transduction insertions. tl and sa read pairs are realigned to ensure correct placement onto the reference genome. additional filtering steps are implemented for removal of low-confidence calls l subfamily assessment to assess which subfamily class drives l insertions as well as l1-mediated transductions, consensus sequences for all full-length primate l1s were constructed from multiple reference elements. to discover l subfamilies driving the transduction, longer contig sequences assembled from short reads were realigned to the primate-specific l consensus sequences. a best mapping criterion was used to infer the most probable l subfamily involved in transduction at each locus. pcr and minion based experimental validations experimental validations of l1-mediated transduction predicted loci were performed using pcr coupled with capillary sequencing, as well as by single molecule long dna read sequencing. human tiger calls were validated using a pacbio whole genome sequencing dataset of na. oxford nanopore minion data was generated based on long-range pcr amplicons, using a sequencing platform that we acquired as part of the minion early-access programme.  computational discovery of l1-mediated transductions through tiger tiger scans genomic regions for the presence of three mobile element transduction-defining signatures: characteristic hallmarks of mobile element insertions, including non-reference polya tails and tsds, which are retrieved from the transposable element analyzer algorithm; aberrant mapping of paired-ends indicative of the adjacent insertion of an additional unique sequence originating from a distal locus; single-anchored paired-end reads that are reassessed by tiger to further substantiate insertion signals. the modular nature of tiger enables it to be applied with any tool for polymorphic mobile element insertion detection. subsequently, tiger pursues additional event characterization steps including realignment, read clustering and filtering to identify high confidence transduction events. to test tiger’s utility for detecting polymorphic l1-mediated 3′ transductions, we applied the tool to fifteen recently published genomes from three non-human primate species. an example transduction event detected by tiger is depicted in fig.  this event involves an inter-chromosomal duplicative insertion of a unique sequence of chimpanzee chromosome into the respective target region mediated by an l1-driven transduction. by realignment onto the reference genome with blat we placed previously unmapped reads onto the reference genome facilitating characterization of the l1-mediated transduction, as visualized in fig. 2a. a more detailed view of read placements is provided in fig. 2b, with one read mapping to the target locus on chromosome and the other read either mapping to a non-reference l element or forming a cluster of reads uniquely mapping to the source on chromosome. some of these latter read mates contain the non-reference polya tail and target site duplication. the additional polya signal, causing termination of the transduced sequence during the transcription process, is also visible in the data. we additionally evaluated the sensitivity of tiger for predicting 3′ l1-mediated germline transductions in ngs data by performed in silico simulations, estimating a sensitivity of 86 %.fig. computational analysis of the chr7:6620368- insertion into the chr10:54643580- region in the chimpanzee sample pr a depiction of the chr10:54643580- region using the integrative genomics viewer before read realignment. after realignment using blat many initially single-anchored reads were placed correctly, facilitating the ascertainment of this l1-mediated transduction clustering to a region on the source chromosome with an average uniqueness of. b a detailed view of l1-mediated 3′ transduction read placements: one read is shown to map to the target locus on chromosome and the other read maps either to a non-reference l element or forms a cluster of reads uniquely mapping to the source on chromosome. out of reads, were carrying parts of a non-reference polya tail experimental verification of transductions by pcr and capillary sequencing to verify the accuracy of tiger, we performed validation experiments on randomly chosen 3′ transduction calls, using pcr followed by capillary sequencing. we employed a combination of an outer and inner primer pair to specifically amplify the target region, and to overcome the barriers brought about by the two respective polya tails for pursuing validation by capillary sequencing. this validation strategy enabled verification of both the presence of the mei and of the transduced unique sequence. a representative pcr gel picture for macaque, using outer primers, is shown in fig. 3b. a circos plot depicting all predicted transductions in macaque is provided in fig. 3c. in total, we verified out of 51 l1-mediated transduction calls, based on which we estimated a false discovery rate of  %. investigation of the experimental data on the eight false positive loci revealed that seven lacked meis as well as the transduced sequence, whereas the remaining locus presented evidence of an l insertion but lacked the inferred transduced sequence.table summary of tiger results in non-human primate species for comparison to na see additional file 1: table s5 *determined based on ratio between tiger transductions and l insertions. 95 % confidence intervals were calculated using one sample t-test **significantly different based on wald test of predicted-transduction rates: chimpanzee-macaque: p = ; chimpanzee-orangutan: p = ; macaque-orangutan: p =  fig. experimental verification of tiger-based l1-mediated 3′ transductions by pcr. a general primer design: outer primers were placed outside of the event in the target locus to amplify the l1-mediated sequence transduction insertion allele and/or the reference genome allele. on the left side of the locus, the corresponding sequence uniquely matches the target site, and subsequently matches to multiple positions in the genome in line with the presence of an l element. further to the right, the sequence will also match uniquely to the target site and end with a polya stretch not seen in the reference genome. in order to confirm the presence and origin of the transduced sequence, we employed a 2nd set of primers inside the predicted unique transduction sequence. b example pcrs verifying rhesus macaque l1-mediated sequence transductions, based on outer primers, are shown for inferred carrier and non-carrier samples. in the presence of an l1-mediated transduction sequence insertion, a larger band than the reference band in nc is seen; heterozygotes show both bands whereas homozygous l1-mediated sequence transduction insertions show only one band. c a circos plot shows the distribution for all inferred rhesus macaque l1-mediated sequence transductions; experimentally validated insertions by pcr and minion single molecule sequencing are depicted in green. arrowheads indicate directionality towards the target locus tiger can be used for estimation of the size of the transduced sequence – however, this capability is strongly dependent on read length and insert-size of the paired-end ngs library, with short ngs reads being only of limited use for detecting the boundaries of the l element’s 3′ and the transduced sequence’s 5′ at target loci. in our analyses, sizes of computationally inferred transduction sequence lengths varied between 90–260 bp in chimpanzee, 74–437 bp in orangutan and 64–361 bp in macaque. this suggests that tiger’s minimal sequence requirement for reliably inferring transductions in illumina sequencing data is ~50–60 bp. verification and characterization of transductions by single molecule sequencing both short ngs reads and sanger sequencing reads do not typically fully span the target locus, which complicates the characterization of transduction events. we reasoned that third generation long-read single molecule dna sequencing technologies may help overcome this challenge, by fully recovering the complete sequence and structure of the combined insertion. hence, we employed both pacific biosciences sequencing and oxford nanopore minion sequencing to obtain further insights into l1-mediated transductions.fig. pacific biosciences and oxford nanopore minion long read verification of l1-mediated transduction insertions. a left panel: alignment dotplot – surrounding reference genome sequence for the human chr4:104210671- region shown on the x-axis; pacbio read on the y-axis: ~1000 bp shift shows presence of insertion. right panel: inspection of the inserted sequence verified the presence of the l element and the transduced sequence including the new polya tail; the new polyadenylation signal is underlined. b dotplot – with reference genome sequence on the x-axis and minion read on the y-axis: ~1200 bp shift shows presence of an insertion. the inserted sequence verified both the presence of an l element and additional transduced sequence including the new polya tail. c alignment of the inserted l sequence to the ~6 kb long l consensus sequence shows that the integrated l is 5′-truncated we first employed tiger to discover transduction events in the human hapmap dna sample na, which enabled us to overlay tiger transduction calls with long dna sequencing read data previously generated by whole genome sequencing using pacbio technology. our analysis of the data showed that long dna reads are indeed valuable for characterization of l transductions. as an example, alignment of a 7 kb long pacbio read from na to a transduction candidate locus on chromosome demonstrated a ~1 kb shift in the alignment, further substantiating the presence of the insertion predicted by tiger. additional inspection of the pacbio sequence allowed us to characterize the structure of the event in more detail. indeed, analysis of the inserted sequence verified the presence of an l 3′ transduction, with a 908 bp 5′-truncated l element exhibiting a 126 bp long transduced sequence ending with a polya tail in 3′. using the previously published pacbio wgs data for na as well as fosmid sequencing data generated previously for na, we verified four out of six l1-mediated transductions identified in this human sample. from the remaining two human events, one showed a solo l insertion upon further inspection of the pacbio reads, whereas the other locus remained inconclusive, as it lacked coverage of pacbio reads at the genomic region in question, preventing us from verifying the element by computational means. second, we obtained similar validation results for all three non-human primate species, through oxford nanopore sequencing data which we generated as part of the minion early-access programme, following long-range pcr amplification of candidate loci. for example, minion reads spanning the rhesus macaque l transduction candidate locus on chromosome verified the presence of a ~  kb insertion, and further analysis of the inserted sequence demonstrated a 116 bp long 5′-truncated l element and transduction of 1043 bp of additional sequence including the new polya tail in 3′. overall, we validated transductions by single molecule sequencing, which combined with the pcr validations resulted in experimentally validated l transductions – to our knowledge the largest dataset on validated non-reference germline mobile element transductions reported to date. facilitated by the generated long read sequencing data we examined the length distribution of the inserted l elements and of the transduced sequences, observing transduction sequence lengths ranging from to 1570 bp. our validation experiments further verified an abundance of 5′ l sequence truncations as previously reported in a similar context. among experimentally validated transductions, most showed relatively small l elements. investigation of transduction rates in primate species we further made use of inferred transduction events to estimate rates of transduction in different non-human primate species, encouraged by earlier studies demonstrating differential activities of solo mobile element insertions across primate species. tiger altogether detected non-redundant l1-mediated 3′ transductions in the primate genomes, in rhesus macaque, in orangutan and in chimpanzee. average numbers of l1-mediated transductions per individual were for macaque, in orangutan and in chimpanzee. to calculate the rate of transductions per species, we divided the number of high confidence tiger transduction calls by the total number of non-reference l insertions identified using tea or tiger. our transduction rate estimates were significantly different between species with estimates of  % ±  ci for chimpanzee,  % ±  for orangutan, and  % ±  for macaque,, chimpanzee-macaque and orangutan-macaque, table 1). we further tested whether the observed difference in transduction rates among species could reflect underlying differences in the efficacy of selection among non-human primates with different effective population sizes. we observed no evidence that selective constraints varied substantially among these primate species, and obtained little evidence for an impact of effective population size on the efficacy of selection. the total amount of l calls in the human na down-sampled genome was – resulting in a transduction rate estimate of ~  %. similarly, filtering the kidd et al. data for calls overlapping with l elements in the target regions and requiring more than 50 bp of uniquely mappable transduced sequence, resulted in an adjusted transduction rate estimate of  %. lastly, we examined whether tiger detected 5′ transductions in our primate dataset. notably, we did not observe evidence for a single l 5′ transduction event driven by a new upstream promoter, which is consistent with earlier reports based on reference transduction analysis suggesting a very low rate of such events. characterization and l subfamily analysis of transduction events to investigate potential functional consequences of transductions we analysed the overlap of transduced sequences with annotated functional regions of the genome, considering all events identified in this study. the majority of transduction sequences were derived from intergenic regions and a similar fraction also inserted into intergenic regions. approximately a third inserted into intronic regions, where some may have an effect on gene regulation. intersection of source coordinates of inferred transductions with exonic annotations, furthermore, identified two candidate events – one in orangutan and one in macaque. in both cases, while there was strong evidence for the insertion of unique genomic sequence, evidence for l associated sequence signatures was minimal. notably, following further manual inspection and pcr validation, both insertions turned out to represent gene retrocopy insertion polymorphisms rather than transduction events. grips share many diagnostic features with transductions, such as a tsd and the insertion of unique sequences, including the presence of a polya tract, as they are mobilized by the l machinery in trans, which may explain why tiger was able to identify these events in this context. we further investigated the source-donor l relationship with a focus on transduced sequences. among three out of the six transduction calls observed in humans, we obtained evidence for a full-length reference l element immediately upstream of the predicted transduction source locus. contrary to the human genome, we saw no clear indication confirming the presence of full length donor l elements at source loci within non-human primates, apart from one source region in rhesus macaque exhibiting a >5 kb long l1pa element within a 10 kb region surrounding the transduced source sequence. we therefore classified the source loci of all validated transductions into two classes: no donor l fragments annotated within 5 kb to either side of the transduced sequence and presence of small, truncated l elements surrounding the transduced sequence, either unrelated to the transduction or severely truncated following the formation of the transduction event. our analysis suggests that the majority of calls belong to class, i.e. in rhesus macaque, in orangutan and five in chimpanzee, whereas the remainder fall into class. in addition, our analysis did not reveal any hotspot donor l1s generating multiple transductions, as recently described in an in-depth analysis of somatic transduction events in cancer genomes. finally, we investigated l subfamilies responsible for transductions to evaluate whether l subfamily specificity may explain the differential rate at which l elements are accompanied by transduced sequences in different species. to this end we remapped contigs assembled from short reads aligning to the inserted l sequence to the consensus l subfamilies and enumerated best alignment hits. notably, 96 % of all annotated l1-mediated transduction insertions in rhesus macaque belonged to the l1cer family, which has evolved from macaque-specific l1pa. in orangutan, by comparison, 63 % of the annotated l1-mediated sequence transductions were associated with the l1pa subfamily and 92 % with any l1pa family member. furthermore, in chimpanzee all transductions were found in association with l1pt members. interestingly, we observed examples where l1s accompanied by transductions showed a different subfamily distribution than solo l1s in the respective species. in macaque, for example, l1cer- showed a slight enrichment for transductions, albeit not nominally significant, whereby l1cer- associated transductions showed depletion relative to solo l1s. by comparison, in orangutan l1pa elements exhibited an increased rate of transductions relative to solo l1s, whereby in chimpanzee too few l transductions with reliable subfamily annotation were identified to allow for robust statistics.fig. l subfamilies associated with l1-mediated transductions: p values are based on fisher’s exact test per subfamily using 2 ×  contingency tables discussion retrotransposon mediated transductions are an important class of polymorphic structural variation in the germline, which so far has been largely unexplored, and tiger renders this class of genetic variation amenable to ngs-based analyses. while the detection of transductions presents technical challenges in short read dna sequencing data, owing to the repetitive nature of mobile elements and the fact that illumina sequencing reads are short when compared with l sequences, we have demonstrated tiger’s ability to robustly identify l1-transduced sequence elements. our data indicate variability in transduction rates between species, with rhesus macaque and chimpanzee exhibiting significantly reduced transduction rates compared to orangutan, which adds to previous findings of differential activities of mobile elements among primate species. we note that a number of l insertions associated with transductions in the samples covered by our study were previously overlooked in a scan for solo l insertions, presumably since in the presence of a transduction these events lacked sufficient evidence from both 5′ and 3′ flanks for solo l inference – which indicates that application of tiger can increase sensitivity for l detection. differences in transduction rate may have evolutionary consequences, given that transductions can mobilize functional genomic dna sequences. these differences may at least in part be mediated by l subfamily usage. our transduction rate estimate for humans is slightly lower when compared to previous studies reporting ~10 %, a difference that may be attributed to the stringent filtering we performed. kidd et al. identified transductions accompanying 20 % of all non-reference l1s, resulting in a higher transduction rate – but when we employed equivalent filters used in conjunction with tiger on the kidd et al. data, we obtained a comparable transduction rate estimate of  %. it should be stressed that we designed tiger for the analysis of short ngs reads, which are known to map ambiguously in the context of repetitive genomic sequence when compared to pacbio or capillary sequencing reads – and remaining limitations related to the use of illumina data exist across methods utilizing short reads. thus tiger may be insensitive to elements that insert into or derive from regions of low mappability, as well as to transductions <50 bp in length. another limitation of our study is that we did not specifically investigate orphan transductions arising from the same process. it is possible that an improved fdr of tiger may be achieved in conjunction with higher confidence mei calls. in this regard, our fdr estimate for tiger is well in line with a recent fdr estimate for the tea mobile element discover algorithm used for inferring mei signals in our study. furthermore, our investigation of pacbio and minion single molecule dna long read sequencing data demonstrates the potential of third generation sequencing for uncovering such events, with the promise to facilitate identification of these also in more repetitive regions of the genome as already suggested in a study by chaisson et al., once such technologies are more widely applied at a genome-wide scale. although tiger should in principle be capable of identifying 3′ and 5′ transductions accompanying l insertions, we have not observed a single 5′ transduction event driven by an alternative upstream promoter in our data. 5′ transduction events were shown to be extremely rare in human genomes with only few cases reported. scarcity of 5′ l transductions may also relate to the common truncation of the 5′ part of the transcript observed during the tprt based integration mechanism, which would be expected to particularly affect 5′ transduction sequences upstream of the l element. a recent study of somatic l transduction events in cancer genomes is consistent with a scarcity of 5′ l transductions. cancer-associated transductions, furthermore, have been reported to be typically highly clustered, whereby a single source l1-master element tends to cause several transduction events in unrelated samples. interestingly, we did not observe such clustering in the samples studied here, perhaps due to relaxed suppression of active l elements in the germline, which may reduce event clustering. in somatic tissues most of active l sources are suppressed, and clustering may occur when one or few escape from the suppression, mediated, for example, by local alterations in chromatin structure and dna methylation in cancer cells. our study focused on the inference of polymorphic l transductions, with l elements belonging to the autonomous retrotransposition-competent mobile element class commonly mobilizing non-repetitive sequences. while sva elements are also capable of transducing unique dna sequences and therefore could be a future scope of further tiger development, they are still absent from the macaque genome, and previously observed novel non-reference sva elements in other species were relatively small in number. alu-mediated transductions have so far not been reported in the literature and thus were not a target of our study. in principle, while we chose to work with the tea algorithm to identify l input signals, tiger can be used in conjunction with other mei discovery algorithms, including tangram, mobster, te-tracker and retroseq, augmenting the functionality of these existing tools. by mobilizing unique sequences, l elements can shuffle and duplicate potentially functional genomic segments adding to genomic diversity. tiger enables investigation of this relevant layer of genetic diversity in personal genomes, with potential future applications to disease and evolutionary studies.  we developed tiger for identifying non-reference retrotransposon-mediated transductions in the germline using ngs data. tiger, which can be used in conjunction with a number of translocation and non-reference retrotransposon discovery tools, will enhance variant analysis pipelines, and offers access to an as yet under-explored type of germline genetic variation. ethics and consent to participate not applicable. consent to publish not applicable. availability of data and materials all data presented in this study is either a part of the manuscript or supplementary files listed below. additional files additional file 1: contains all supplemental material and methods and supplemental figures and tables. additional file 2: is a list of all l1-transductions discovered in this study and their validation status in macaque, orangutan, chimpanzee and human. additional file 3: contains a list of all l1-transductions discovered in this study near genes that either originate from an intronic sequence or insert into an intronic sequence in macaque, orangutan, chimpanzee and human. additional file 4: contains igv representations from each experimentally validated source locus. abbreviations fdrfalse discovery rate gripgene retrocopy insertion polymorphism memobile element meimobile element insertion ngsnext generation sequencing pa/polyapolyadenylation sasingle-anchored svstructural variant tltranslocation tstransduced sequence tsdtarget-site duplication competing interests the authors declare that they have no competing interests. authors’ contributions jt, ams and jok designed the study. jt developed tiger. el and pjp performed l detection and subfamily analyses. og, au and ams performed validation experiments or validation experiment analysis. sm designed and executed in silico transduction simulations. dg and eemf provided population genetics analyses. jt, el, ams and jok made contributions to writing the manuscript, with additional input from all remaining authors. all authors read and approved the final manuscript. 
we employed tiger to characterize polymorphic transductions in fifteen genomes from non-human primate species, as well as in a human genome. we achieved high accuracy as confirmed by pcr and two single molecule dna sequencing techniques, and uncovered differences in relative rates of transduction between primate species. by enabling detection of polymorphic transductions, tiger makes this form of relevant structural variation amenable for population and personal genome analysis.
 while active line- elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking.  here we present the computational approach tiger, enabling the discovery of non-reference l1-mediated transductions by combining l discovery with detection of unique insertion sequences and detailed characterization of insertion sites. we employed tiger to characterize polymorphic transductions in fifteen genomes from non-human primate species, as well as in a human genome. we achieved high accuracy as confirmed by pcr and two single molecule dna sequencing techniques, and uncovered differences in relative rates of transduction between primate species.  by enabling detection of polymorphic transductions, tiger makes this form of relevant structural variation amenable for population and personal genome analysis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords retrotransposonl1transductionsngsbioinformaticsgenomegeneticsprimatessingle-molecule sequencingemmy noether grantko4037/1-1issue-copyright-statement© the author 2016 