BACKGROUND
insertional mutagenesis is a widely used and powerful technique to discover and study the function of genes in most organisms.
in pathogenic microbial organisms mutagenesis has played a pivotal role in early gene discovery and is still used alongside more targeted gene deletion approaches for gene function analysis.
in pathogenic fungi the methods applied include random marker gene insertion using plasmids  <cit> , agrobacterium tumefaciens transformation   <cit>  and transposon mutagenesis  <cit> .
these forward genetic methods aim to be relatively unbiased, but hotspots of insertion events are sometimes reported  <cit> .
mutants are generated and tagged by nucleotide changes or by the integration of a transgenic marker gene into critical promoter regions or into protein encoding sequences.
the affected genes frequently show altered gene expression and/or the generation of truncated proteins.
in mutant screens, typically hundreds or thousands of mutants are generated and screened for a desired alteration in a specific phenotype.
for filamentous haploid plant pathogens with moderate sized genomes typically the loss of pathogenicity is tested in glasshouse or controlled environment based screens using the most suitable host plant and bioassay.
once relevant mutants are identified, tagged gene are isolated using a variety of methods.
these methods include the use of genetic maps and genome walking  <cit> , polymerase chain reaction  based approaches  <cit>  or plasmid rescue  <cit> .
more recently the introduction of affordable high-throughput sequencing techniques has facilitated the analysis of whole genomes  <cit> .
these methods include the currently widely used illumina sequencing-by-synthesis approach with fluorescently labelled reversible-terminator nucleotides  <cit> .
the availability of reference genomic information for many species allows the characterisation of mutants using whole-genome sequencing .
such an approach was earlier reported for bacterial and protozoa species of small genome sizes up to 23 mb to identify transposon insertion mutations  <cit> .
also for two saprophytic ascomycete model fungi of 40 mb genome size, wgs was shown to be suitable for the identification of developmental genes and narrowing down of candidate mutations induced by chemical, uv and γ-radiation  <cit> .
however for pathogenic filamentous fungi this technology is not successfully established.
previous wgs approaches have been hindered by a combination of factors.
these factors include, poorly designed methodologies, suboptimal sequence read length, the library size used during sequencing and the presence of high amounts of repetitive dna in a range of microbial pathogens such as magnaporthe oryzae .
fusarium graminearum  is a globally important pathogen of wheat, maize and barley.
the pathogen belongs to the filamentous ascomycetes and has a genome of 37 mb in length, distributed over predominantly four chromosomes.
a high quality genome assembly exists and this is aligned to a genetic map  <cit> .
f. graminearum is a haploid organism and effective transformation techniques are established  <cit> .
a natural gene silencing mechanism called repeat induced point  mutation exists in f. graminearum which mutates duplicated sequences  <cit> .
hence inspection of the genomic dna sequence revealed the presence of only a minimal amount of repetitive dna.
f. graminearum therefore appears to be ideally suited to test a whole-genome sequencing approach for insertion mutant characterisation.
in this study we explored a whole-genome sequencing approach using the illumina hiseq  <dig> platform to identify insertion points and possible genomic rearrangements in four selected plasmid insertion mutants with reduced virulence to wheat.
the analysed mutants included low, medium and high-complexity insertion events, pre-screened using southern  analysis.
we set up a de novo bioinformatics workflow called findinsertseq.
the findinsertseq bioinformatic pipeline successfully identified dna tag insertion sites with a precision to the nucleotide level for the four mutants analysed.
subsequent sequenced read coverage inter-comparisons of the mutant genomes revealed additional recombination events including larger tagged and untagged chromosome deletions, and dna amplification events.
using a random data sampling approach, the minimal genome coverage required for the findinsertseq workflow to provide reliable results was determined to be 22× fold.
we propose that wgs and the findinsertseq approach is an effective method to characterise insertional mutants and transgenic strains expressing constructs of interest in filamentous fungi.
RESULTS
isolation of reduced virulence f. graminearum mutants with insertion events of different complexities
an insertional mutagenesis screen of the f. graminearum wild-type strain ph- <dig> was completed using a linearised plasmid vector containing the hygromycin phosphotransferase gene under control of the trpc promoter.
in total,  <dig> stable transformants were generated and screened on wheat ears of the cultivar bobwhite for reduced virulence using previously described methods  <cit> .
eight mutants that showed highly reduced virulence were identified .
insertion events were characterised using southern blot analysis and three of the disease attenuated fusarium mutants, called daf <dig>  daf <dig> and daf <dig> were chosen for analysis in this study .
a fragment of the hph phophotransferase resistance gene was used as a probe.
the mutagenesis plasmid phyg <dig>  has single restriction sites for ecori and xhoi .
for daf <dig>  genomic dna was cut with both enzymes to produce single hybridisation bands indicating a single insertion event.
for daf <dig> the xhoi digest revealed two fragments at  <dig>  and 5 kb consistent with the insertion of more than one possibly rearranged plasmid.
for daf <dig> the presence of multiple hybridisation fragments of different sizes indicated a highly complex insertion event.
the fourth mutant explored as a control in this study was the previously characterised non-virulent f. graminearum strain tp <dig> .
this strain was previously demonstrated in a targeted pcr based analysis to harbour a single insertion event in the top <dig> gene  and was generated using the same plasmid  <cit> .figure  <dig>
dna blot analysis of three mutants showing low, medium and complex insertion events.
daf mutant genomic dna was digested with two restriction enzymes and probed with a radioactive labelled hph gene.
lanes  <dig> to 3: ecori digested dna of daf <dig>  daf <dig> and daf <dig>  lanes  <dig> to 6: xhoi digested dna of daf <dig>  daf <dig>  daf <dig>
sequencing and findinsertseq bioinformatics approach to identify insertion tags and transformation-induced mutations
illumina sequencing was used to obtain high whole-genome coverage for the three daf mutants and the control strain tp <dig> .
genome coverage of  <dig> to  <dig> times was achieved by 100-bp paired-end reads from inserts with a mean library fragment size of 430 bp on an illumina hiseq  <dig> system .
to detect the plasmid insertion sites an analysis pipeline, called findinsertseq, was devised .
the 5′- and 3′-mate sequences of the paired-end reads were mapped to the plasmid reference sequence.
the pairs where both mates did not map to the plasmid were discarded.
the pairs where one mate mapped to the plasmid and the other mate did not map to it, were presumed to be located at the border of the plasmid and the fusarium genomic dna.
from these desirable sequences, the latter of the pair was extracted.
in these pairs the unmapped mate represented either the forward or reverse sequences that corresponded to either side of the inserted plasmid sequence .
these extracted unmapped mate reads were then mapped to the f. graminearum reference genome, already available for the strain ph- <dig>  resulting in two stacks of reads for each plasmid sequence insertion.
for the f. graminearum strain tp <dig>  with a single plasmid insertion site in chromosome  <dig>  sequence read stacks for the two plasmid flanks are displayed .
the identification of loci marked by the two read stacks was achieved by sorting the mapped reads by base pair position in the sequence alignment map  file.
then a python script using a sliding window of 300 bp to count mapped reads was used to identify where the read coverage peaked.
the peak identified represents both the forward and reverse stacks and is reported by the start and stop base pair positions of the 300-bp window and the chromosome number .
for mutants of interest, confirmation of the flanking regions reported by the pipeline and any deletion of reference sequence via the gap in coverage between these flanks was verified using a genome browser software such as tablet  <cit> .
this was used in combination with a binary alignment map  file generated by mapping the unmapped mates from the plasmid to the ph- <dig> reference.
however, in genome regions where no reference sequence was available, or where no read stacks were found suggesting that the genomic reference was incomplete another approach was required.
the extracted reads were de novo assembled to compile the genomic sequences flanking the inserted plasmid.
this later approach was required to resolve the insertions sites of mutants daf <dig> and daf <dig> .figure  <dig>
findinsertseq: sequencing workflow to identify dna tag insertion sites and corresponding flanking genomic sequences.
step  <dig>  paired-end reads are mapped to the plasmid reference sequence, step  <dig>  extract unmapped mate reads from the paired partners that should contain sequences either side of the inserted plasmid sequence.
map the extracted mate reads to the f. graminearum reference genome which should form two stacks for each plasmid sequence insertion with one in the forward  orientation which are mated with the reads that originally mapped to the plasmid at the 5′-end and a stack with reverse  orientated matching reads originally mapped to the plasmid at the 3′ end.
step  <dig> a) the availability of reference genome is a prerequisite for this workflow.
b) * if no reference genome is available de novo assembly of reads to identify flanking regions could be considered followed by blast and pcr analysis to characterise the insertion site.
step  <dig>  to locate the stack chromosome and bp positions, the mapped reads are sorted, positioned and printed to the command line.
additionally the results are printed to an individual sample text file.
open tablet software  <cit>  with the mapped genomic reference bam file and proceed to the chromosome and position that were identified in step  <dig> to visualise the insert flanking sequences and any deletion of reference sequence via the gap in coverage between the two stacks.
when a reference genome assembly is either not available or both flanking sequences have not been detected due to the local incompleteness of the reference genome, then de novo assembly of the extracted unmapped mate reads can be used to reconstruct the sequences replacing steps  <dig> a through  <dig> with step  <dig> b.
visualisation of a single insertion site in
f. graminearum
strain tp <dig> .
sequence reads are visualised in a screenshot using tablet software  <cit> .
sequence reads mapped to this region of the genome,  coloured lines represent the genomic sequence of the visualised region and frame translations,  sequence coverage histogram, and  mapped reads for both flanking regions and the narrow gap between the two read stacks which marks the position of the plasmid insertion.
forward green stacks and reverse purple stacks are shown.
mapping the insertions
findinsertseq localised the insertions in chromosome  <dig> for strain daf <dig> and daf <dig> and chromosome  <dig> for daf <dig> and tp <dig>  .
for daf <dig>  the tag insertion into chromosome  <dig> was accompanied with a small 12-bp deletion.
for tp <dig> , our wgs analysis confirmed the result obtained by baldwin et al.
<cit>  that the plasmid insertion into chromosome  <dig> caused a 4-bp deletion.
tag insertion in daf <dig> and daf <dig> were accompanied with more extensive deletions next to the insertion sites .
for daf <dig>  the insertion occurred downstream of a telomeric sequence repeat   <cit> .table  <dig>
location of insertion tags and deletion events at the insertion site
isolate
chromosome location
sequence at the 5′ end of the insertion
supercontig sequence deletion*
sequence at the 3′ end of the insertion
plasmid insertion site
taaccc
†
not identified
*where the deletion is large, bp positions are given.
†telomere sequence taaccc given in italics.
for three of the strains the plasmid insert positions were established by mapping to the plasmid and subsequent mapping of mate reads to the reference genome.
whereas for daf <dig> de novo assembly was necessary to identify the sequence at the 5′ end of the insertion due to an incomplete reference sequence in this part of the f. graminearum genome.
the 5′-flanking insertion sequence was not identified for daf <dig> using either approach.
identification of chromosome deletions and amplifications
the sequencing of the mutant genomes permits further qualitative analysis which may be missed using molecular biology methodologies resulting in falsely linking a retrieved gene sequence to a phenotype.
for each of the daf mutants, the obtained genomic reads were mapped to the f. graminearum reference genome .
this analysis detected additional deletion and/or copy number variations of genomic sequence associated with the plasmid insertion process.
other formal possibilities by which variation could have been introduced are cellular stress responses induced by the transformation method, growth and storage conditions.
in the tp <dig>  mutant no detectable amplifications or deletions other than at the plasmid insertion site were detected.
for daf <dig>  the dna gel blot analysis had predicted a single plasmid insertion event into the genomic dna .
however, this further sequence analysis revealed that besides the insertion in chromosome  <dig>  an additional untagged chromosomal deletion had occurred.
this untagged deletion event had resulted in the loss of  <dig>  bp at the 3′-end of chromosome  <dig> predicted to contain  <dig> genes .
for daf <dig>  the dna gel blot analysis suggested the presence of potentially two copies of the plasmid .
the wgs analysis revealed that the single insertion site was accompanied with a  <dig> -kb deletion close to the 5′-end of chromosome  <dig> resulting in the loss of  <dig> genes upstream.
however, further inspection of the daf <dig> wgs data also identified a twofold amplification of  <dig>  mb immediately downstream of the deletion event .
for daf <dig>  the dna gel blot analysis had suggested a highly complex insertion event.
the plasmid insert in daf <dig> resulted in the loss of  <dig>  kb at the 5′-end of chromosome  <dig> but had left the telomere intact.
the deleted genomic region in daf <dig> is predicted to contain  <dig> genes .figure  <dig>
schematic overview of chromosomal recombination and deletion events for the three daf mutants and verification by diagnostic pcr.
daf <dig> partial chromosomal deletion and insertion event.
for comparison the chromosomal deletion for mutant daf <dig> published earlier by baldwin et al.
<cit>  is indicated.
pcr analysis of chromosomal regions predicted to have either been lost or retained on chromosome  <dig> in strain daf <dig>  the wild type and daf <dig> genomic dna was amplified with oligonucleotide pairs in lanes:  u516/u <dig>   u518/ <dig>   u550/u <dig>   u549/u <dig>   u606/u <dig>   u520/u <dig> and  u522/u <dig>  the approximate location of the amplified pcr fragments is indicated in the schematic overview from left to right corresponding to pcr products in lane  <dig> to  <dig>   daf <dig> shows a single-copy insertion in chromosome  <dig> and a large untagged deletion on chromosome  <dig>   diagnostic pcr in lanes:  u533/u <dig>   u553/u <dig>   u603/u <dig>   u535/u <dig>  approximate location of fragments is indicated in the schematic from left  to right .
diagnostic pcr of wild type and daf <dig> chromosome  <dig> in lanes:  u540/u <dig>   u541/u <dig> and  u556/u <dig>  approximate location of fragments is indicated in the daf <dig> schematic from left  to right .
in daf <dig> three multimerised plasmids are inserted at position  <dig>  bp in chromosome  <dig>   diagnostic pcr of wild type and daf <dig> chromosome  <dig> in lanes:  u600/u <dig>   u558/u <dig>   u545/u <dig>   u545/u <dig>   u604/u <dig> and  u547/u <dig>  approximate location of fragments is indicated in the schematic from left  to right .
large black lines indicate the chromosomes .
chromosomal deletions , plasmid insertion point .
diagnostic pcr fragments .
dna ladder bsteii .
sequence coverage line graphs of characteristic differences at the genome level for the three daf mutants compared to the control strain ph-1
top-1
.
average numbers of sequence reads per  <dig> bp interval were mapped to the wild type reference genome.
top panel, daf <dig> partial chromosome  <dig> deletion event.
middle panel, daf <dig> partial chromosome two deletion event .
bottom panel, daf <dig> partial chromosome four deletion event and twofold amplification of a  <dig>  mb region  downstream of the deletion event.
the open triangle  in each daf mutant plot indicates the position of the loss in genomic sequence.
for each mutant, the corresponding chromosome in the ph- <dig> top <dig> mutant  is depicted as a control.
deletion of gene regions in the four mutants and numbers of genes lost
strain
chromosome
position of sequence loss
number of genes contained within the lost regions
*for daf <dig>  the plasmid vector insertion occurred in chromosome  <dig> .
chromosome  <dig> was affected by an untagged gene deletion event.
estimation of plasmid copy number
for two of the daf mutants, daf  <dig> and daf <dig>  the plasmid copy number inferred from the dna gel blot analysis and the number of insertion sites detected within the f. graminearum genome by wgs analysis did not precisely match.
therefore an estimation of plasmid copy number by wgs was attempted for all four mutants.
to achieve this, the coverage histograms of tp <dig>  and the three daf mutants were plotted across the whole plasmid sequence of the vector phyg <dig>   to identify whether there had been recombination of the plasmid resulting in incomplete plasmid sequence insertion.
average calculated genome wide coverage for the tp <dig>  strain was  <dig>  vs. the experimental observed value of  <dig> for the mapped reads to the plasmid sequence.
this suggested an approximate  <dig> × plasmid length insertion in tp <dig> , however only 1× plasmid length insertion was detected from the de novo assembly.
this result suggests that this wgs–plasmid alignment approach for the estimation of plasmid copy number is fairly accurate.figure  <dig>
coverage histogram of reads mapped to the inserted plasmid in the four mutants.
rows are:  tp <dig>   daf <dig>  daf <dig> and  daf <dig>  the experimentally obtained plasmid average read coverage x¯ for each mutant is given on the far right.
the reference genome read coverage for tp <dig>  is  <dig> .
row  <dig> depicts the genetic elements of the insertion plasmid.
trpc promoter,  hygromycin-phosphotransferase,  f <dig> origin of replication,  ampicillin and
e. coli oric.
coverage of the three daf strains across the plasmid sequence was found to be variable with either some segments absent and/or other segments with increased read density indicating multiple copies of plasmid sequence at the insertion site.
in daf <dig> the plasmid had an average observed coverage of  <dig> suggesting an insertion sequence length of approximately  <dig> plasmids based upon a calculated read-coverage of  <dig>  however some segments of the sequence were over represented.
a similar case was found for sample daf <dig> with an average coverage of  <dig> but a calculated coverage of  <dig> suggesting a length of three plasmids inserted into the genome.
pcr amplification of the concatenated plasmid is hindered by the large size of the concatemer and due to the fact that it consists of repeated elements.
similarly, de novo assembly is hampered due to the repetitive nature of the concatemer that had formed.
the data obtained for daf <dig> suggests a single plasmid insertion of 50% of the length of the sequence with an observed coverage of  <dig> in the regions where coverage was evident vs. a calculated coverage of  <dig>  both tp <dig>  and daf <dig> insertion sequences were validated using de novo assembly and support the reported findings.
interestingly, the plasmid gene cassettes noticeably amplified in daf  <dig>  daf <dig> and daf <dig> include the resistance marker hygromycin-phosphotransferase and the bacterial ampicillin resistance gene while the e. coli oric replication sequences of the plasmid vector were found to be less frequently amplified.
collectively, these data indicate that multiple plasmid concatenations at a single insertion site had occurred in both the daf <dig> and daf <dig> mutants.
experimental verification of insertions and chromosomal deletions by pcr
to demonstrate that the computationally predicted plasmid insertion sites can be verified in the laboratory, insertions and chromosomal deletions were confirmed using a pcr based approach.
this test also excluded the possibility of translocations of affected chromosomal regions into other chromosomes.
primers were designed to detect the presence or absence of ~ <dig> -kb diagnostic fragments across the modified chromosomes.
plasmid insertion sites were verified by designing oligomer pairs where one primer site lies within the inserted plasmid and the corresponding primer site lies within the adjacent chromosomal neighbourhood.
for daf <dig>  four diagnostic fragments located in the region of chromosome  <dig> predicted to be deleted, could not be amplified while amplification of wild type  dna produced the expected fragments .
two further diagnostic fragments upstream of deleted chromosomal region were amplified from both daf <dig> and wt dna.
a diagnostic fragment originating from a primer site located in the inserted plasmid and from the adjacent chromosomal neighbourhood was only amplified in daf <dig> but not in wt dna.
the results verified the computationally predicted chromosome modifications.
for daf <dig> a similar approach confirmed the plasmid insertion site in chromosome  <dig>  in this mutant whole-genome analysis had also revealed an untagged chromosomal deletion in chromosome  <dig>  the deletion and its extent were confirmed using three diagnostic fragments.
for daf <dig> plasmid insertion and a chromosomal deletion was confirmed for chromosome  <dig>  the exact plasmid insertion sites were further confirmed by sequencing the diagnostic fragments obtained using the plasmid specific oligomers.
these data independently confirm the insertion and chromosomal deletion results predicted using our whole-genome sequencing and bioinformatics data analysis pipeline described in this study.
virulence genes identified close to the insertion points or in chromosomal deletions
the adjacent gene close to the plasmid insertion points and genes contained in the chromosomal regions deleted in mutants daf <dig>  daf <dig> and daf <dig> were analysed by blast analysis against the pathogen-host interactions  database  composed of experimentally verified, virulence and effector proteins from bacteria, fungi and oomycete pathogens that infect a wide range of hosts  <cit> .
phi-base associates gene and protein information with published fungal mutant phenotypes.
these phenotypes include altered virulence phenotypes , unaffected pathogenicity, and lethal .
in the deleted regions of daf <dig>  daf <dig>  daf <dig> the blast analysis revealed the presence of  <dig>   <dig> and  <dig> phi-base homologs with the phenotype “altered virulence” at e ≤  <dig>  .
we conclude that the absence of these functional homologs of virulence-associated genes is responsible for the pathogenicity defect of the three mutants on wheat.
the blast analysis also revealed that in the three mutants a total of five putative transcription factor genes had been deleted that were previously implicated to be essential genes in f. graminearum.
in a genome-wide single gene deletion study of transcription factors no successful reverse genetics mutants could be obtained  <cit> .
these genes are in daf <dig>  fgsg_ <dig> , in daf <dig>  fgsg_ <dig> , fgsg_ <dig>  and in daf <dig>  fgsg_ <dig> , fgsg_ <dig> .
the absence of these transcription factor genes was confirmed by pcr amplification using the wild-type strain ph- <dig> as the positive control .
we conclude that the five transcription factor genes are not required for growth in the characterised f. graminearum insertion mutants generated in the strain ph- <dig>
random sampling
subsampling of the illumina sequence read dataset was computationally performed for the tp <dig>  mutant strain using three different seed values.
the original data set had an approximate coverage of  <dig>  the seqtk program was used to produce three technical replicates for 10% , 25% , and 50%  of the total reads.
the average genome coverage for each data set is  <dig>   <dig>  and  <dig>  respectively.
visual inspection of the datasets  identifies that the insertion tag can unambiguously be identified in all three replicates for the 25% and 50% subsamples but not for the 10% subsample.
we conclude that a 22× coverage obtained by the illumina highseq  <dig> sequencing platform is sufficient for f. graminearum to identify insertion tags.figure  <dig>
random data sampling to determine the minimal genome coverage required for insertion point characterisation in
f. graminearum
strain tp11
.
<dig>   sampling  <dig> with three reduced data replicates of 10% each.
the deletion sequence between 5′ and 3′ read stacks is represented with an arrow in aa.
sampling  <dig> replicates with 25% of data.
sampling  <dig> replicates with 50% of data.
the average coverage for each data set in a, b and c at the insertion region is  <dig>   <dig>  and  <dig> respectively.
the original data set had an approximate coverage of  <dig>
discussion
in this study we setup and describe a bioinformatics workflow called findinsertseq  to efficiently characterise forward genetics mutants created by plasmid insertion in the filamentous plant pathogen fusarium graminearum using illumina whole-genome sequencing approach with paired-end reads.
the four mutants selected showed a reduced virulence phenotype on their host wheat.
the mutants were selected to include low and complex insertion events visualised by dna blot analysis.
using the findinsertseq pipeline we were able to rapidly identify dna tag insertion points to the nucleotide level for the control strain tp <dig>  and three novel disease attenuated fusarium  mutants generated for this study.
the principle of the wgs-findinsertseq analysis approach is to map sequence reads to an insertion vector and use the mate that aligns to the genomic reads to identify the insertion point in the genome.
findinsertseq can also be used to discriminate between single insertion and complex tandem insertion events in a mutant where a reference sequence is available for the species or could be adapted where a reference genome is lacking .
thirdly, when de novo mapped read coverage comparisons of mutant and wildtype dna were included in the method, this revealed the existence of further unexpected recombination events, such as chromosomal deletions and amplifications.
to our knowledge this study, for the first time, demonstrates the analyses of complex insertion events on the whole-genome level in a pathogenic filamentous fungus of medium 37-mb-genome size.
the predictions made using the findinsertseq pipeline were independently confirmed through diagnostic pcr analysis.
current laboratory-based methods to determine insertion points such as tail-pcr  <cit>  and plasmid rescue  <cit>  can, in most cases, only successfully be used to identify the insertion event where a single copy of the insertion tag has occurred.
many research groups over the past 20 years have created mutant libraries of which only a small number of single-copy insertion mutants were successfully analysed  <cit> .
such insertion mutant collections are now amenable for further analysis using wgs analysis and the findinsertseq pipeline.
for sequencing we used the illumina hiseq <dig> platform, which is currently the standard high-throughput sequencing method  <cit> .
sequencing of an 800-bp fragment library using 100-bp paired-end reads generated ~100× fold f. graminearum genome coverage when four samples were pooled across one sequencing lane.
the size of the fragment library is an important factor.
if the fragment size exceeds the insertion vector length, an offset of the insertion point will occur.
this offset can be calculated as mean library fragment length minus insertion vector length.
the choice of mapping software can influence the success of the workflow.
a comparison of novoalign  <cit> , bowtie <dig>  <cit>  and bwa  <cit>  mappers using the default settings showed that only the first two successfully mapped the insert .
however, the bwa aligner did not map the reads to the correct position in three of the four strains analysed.
this result was attributed to the loss of direction of the paired reads because both stacks of the unmapped mate reads were represented in the forward orientation.
an alternative strategy was considered via de novo genome assembly followed by applying blast to identify the insertion site of the plasmid.
however such an assembly-based approach was not implemented due to the higher computational resources required to assemble each mutant compared to mapping first to a short plasmid sequence, followed by aligning a small subset of reads to the reference genome.
for species with larger genomes i.e.
plants, plant-infecting insects or pathogenic protists de novo assembly is challenging and often dependent on many non-intuitive parameters such as k-mer value selection.
in addition the output from the mapping approach  provides three visually compelling features that the insertion site has been correctly identified.
the characteristic features are: two flanks with a similar number of aligned reads separated by a clear gap where the plasmid inserted and deleted a variable number of nucleotides.
the findinsertseq workflow currently advises visual inspection using the tablet software  <cit>  as an optional step for mutants of further interest, to inspect forward and reverse sequence stacks either side of the insertion point  and identify the deletion and flank sequence to 1 bp.
currently only an approximate location of the insertion is given for screening purposes of the first and last base of the highest read coverage peak using a set window size .
this location is written to output  but surmises the region of the genome in which the insertion took place.
this approach permits the biologist to experimentally confirm the insertion point using a pcr-based analysis.
randomly down-sizing the sampled genomic read coverage for the control mutant strain tp <dig>  revealed that only  <dig> x genomic coverage is required to identify the insertion site with a cost incurred per sample of approximately $ <dig> including all associated cost such as dna preparation and library construction.
sequencing methods are evolving rapidly and as a consequence associated costs are expected to drop even further.
many fungal genomes are of similar size to f. graminearum and have a similar gc content.
a wgs-findinsertseq approach can identify multiple locations of plasmid insertions.
however, this may prove more difficult to apply in fungal eukaryotes such as magnaporthe oryzae for which  <dig> % repetitive dna is reported  <cit> .
if the insertion event flanking sequence contains repeat sequences, mapping will be ambiguous.
transformation events may result in reshuffling and partial amplification of the insertion tag  <cit> .
we found this strikingly to be the case for the mutant strain daf <dig>  the coverage histogram  revealed that the resistance marker gene encoding hygromycin-phosphotransferase  was highly amplified during vector plasmid integration as well as the ampicillin gene cassette.
these events could be favoured by dna structural elements in the vector influencing the outcome of recombination events.
the amplification of the hph gene is likely to be selected for when growing the transformants on hygromycin-containing medium.
in addition to the pipeline described in figure  <dig> we investigated situations where only a single flanking sequence was identified.
this outcome suggested further genomic alterations might have occurred.
in these cases all genomic reads from the mutant were mapped back to the reference.
this approach successfully revealed additional tagged and untagged chromosomal deletions and amplifications events in the mutants .
the extent of the chromosomal deletions is surprising and suggests that larger parts of the f. graminearum genome are not required for growth in vitro and / or during the protoplast transformation and regeneration procedure used in this study.
similar results were found for two f. graminearum mutants, called z43r <dig> and daf <dig>  where the occurrence of large chromosomal deletion events was reported earlier  <cit> .
in both studies a combination of plasmid-rescue and sanger single-read sequencing was used to characterise the mutants.
in z43r <dig> a genomic region of 220 kb containing at least  <dig> genes was deleted, whilst in daf <dig> a genomic region of 350 kb containing  <dig> genes was deleted.
the molecular analysis of z43r <dig> revealed a deletion of a genomic region at the vector insertion site, including the gene clusters required for the biosynthesis of aurofusarin and zearalenone.
the authors deliberately recreated the deletion event using a reverse genetics approach to demonstrate that a large region of genomic dna can be efficiently deleted in f. graminearum by double homologous recombination  <cit> .
in the daf <dig> study, the unintentional deletion of a large region of genomic dna permitted the authors to report that the loss of the tri <dig> gene involved in mycotoxin biosynthesis was responsible for the virulence defect on its host.
interestingly, the mutant daf <dig> recreated the daf <dig> mutant and extended the deleted region by an additional  <dig> genes .
taken together, these findings show that large chromosomal regions can systematically be deleted in f. graminearum to understand genes sets and genome organisation.
in s. cerevisiae, such an approach is currently being used to build a minimalist yeast genome within the sc <dig>  project  <cit> .
the deleted chromosomal regions in the analysed mutants identified a total of  <dig> genes that when deleted from the f. graminearum genome are not required for life and are thus unlikely to make good fungicide targets.
within this gene set were five putative transcription factors that were earlier proposed to be essential for life in f. graminearum  <cit> .
son and colleagues  <cit>  tested in a genome-wide gene deletion study  <dig> putative transcription factors, but no gene deletion mutants could be obtained for these five genes.
while it is possible that the absence of these proteins is compensated by the absence of additional proteins within the deleted chromosomal regions, further experiments are needed to elucidate their function.
CONCLUSIONS
this study demonstrates that whole-genome sequence analysis is an efficient tool to characterise forward genetics mutants in the filamentous pathogenic species f. graminearum.
surprisingly in some cases the dna tag insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed.
this fact suggests that the findinsertseq method identifies dna tag insertion points and other genomic recombination events such as chromosomal deletions and dna amplifications.
large scale chromosomal deletions can quickly identify the non-essential gene set or host-specificity genes not essential for pathogenic life.
the minimal sequencing coverage required in f. graminearum to survey accurately the genome to a single base level for recombination events was determined to be 22× fold.
findinsertseq will also allow full characterisation of recombinant filamentous fungal isolates overexpressing heterologous proteins for research and/or industrial scale commercial applications.

 insertional mutagenesis is a widely used and powerful technique to discover and study the function of genes in most organisms. in pathogenic microbial organisms mutagenesis has played a pivotal role in early gene discovery and is still used alongside more targeted gene deletion approaches for gene function analysis. in pathogenic fungi the methods applied include random marker gene insertion using plasmids, agrobacterium tumefaciens transformation and transposon mutagenesis. these forward genetic methods aim to be relatively unbiased, but hotspots of insertion events are sometimes reported. mutants are generated and tagged by nucleotide changes or by the integration of a transgenic marker gene into critical promoter regions or into protein encoding sequences. the affected genes frequently show altered gene expression and/or the generation of truncated proteins. in mutant screens, typically hundreds or thousands of mutants are generated and screened for a desired alteration in a specific phenotype. for filamentous haploid plant pathogens with moderate sized genomes typically the loss of pathogenicity is tested in glasshouse or controlled environment based screens using the most suitable host plant and bioassay. once relevant mutants are identified, tagged gene are isolated using a variety of methods. these methods include the use of genetic maps and genome walking, polymerase chain reaction based approaches or plasmid rescue. more recently the introduction of affordable high-throughput sequencing techniques has facilitated the analysis of whole genomes. these methods include the currently widely used illumina sequencing-by-synthesis approach with fluorescently labelled reversible-terminator nucleotides. the availability of reference genomic information for many species allows the characterisation of mutants using whole-genome sequencing. such an approach was earlier reported for bacterial and protozoa species of small genome sizes up to 23 mb to identify transposon insertion mutations. also for two saprophytic ascomycete model fungi of 40 mb genome size, wgs was shown to be suitable for the identification of developmental genes and narrowing down of candidate mutations induced by chemical, uv and γ-radiation. however for pathogenic filamentous fungi this technology is not successfully established. previous wgs approaches have been hindered by a combination of factors. these factors include, poorly designed methodologies, suboptimal sequence read length, the library size used during sequencing and the presence of high amounts of repetitive dna in a range of microbial pathogens such as magnaporthe oryzae. fusarium graminearum is a globally important pathogen of wheat, maize and barley. the pathogen belongs to the filamentous ascomycetes and has a genome of 37 mb in length, distributed over predominantly four chromosomes. a high quality genome assembly exists and this is aligned to a genetic map. f. graminearum is a haploid organism and effective transformation techniques are established. a natural gene silencing mechanism called repeat induced point mutation exists in f. graminearum which mutates duplicated sequences. hence inspection of the genomic dna sequence revealed the presence of only a minimal amount of repetitive dna. f. graminearum therefore appears to be ideally suited to test a whole-genome sequencing approach for insertion mutant characterisation. in this study we explored a whole-genome sequencing approach using the illumina hiseq platform to identify insertion points and possible genomic rearrangements in four selected plasmid insertion mutants with reduced virulence to wheat. the analysed mutants included low, medium and high-complexity insertion events, pre-screened using southern analysis. we set up a de novo bioinformatics workflow called findinsertseq. the findinsertseq bioinformatic pipeline successfully identified dna tag insertion sites with a precision to the nucleotide level for the four mutants analysed. subsequent sequenced read coverage inter-comparisons of the mutant genomes revealed additional recombination events including larger tagged and untagged chromosome deletions, and dna amplification events. using a random data sampling approach, the minimal genome coverage required for the findinsertseq workflow to provide reliable results was determined to be 22× fold. we propose that wgs and the findinsertseq approach is an effective method to characterise insertional mutants and transgenic strains expressing constructs of interest in filamentous fungi.  isolation of reduced virulence f. graminearum mutants with insertion events of different complexities an insertional mutagenesis screen of the f. graminearum wild-type strain ph- was completed using a linearised plasmid vector containing the hygromycin phosphotransferase gene under control of the trpc promoter. in total, stable transformants were generated and screened on wheat ears of the cultivar bobwhite for reduced virulence using previously described methods. eight mutants that showed highly reduced virulence were identified. insertion events were characterised using southern blot analysis and three of the disease attenuated fusarium mutants, called daf daf and daf were chosen for analysis in this study. a fragment of the hph phophotransferase resistance gene was used as a probe. the mutagenesis plasmid phyg has single restriction sites for ecori and xhoi. for daf genomic dna was cut with both enzymes to produce single hybridisation bands indicating a single insertion event. for daf the xhoi digest revealed two fragments at and 5 kb consistent with the insertion of more than one possibly rearranged plasmid. for daf the presence of multiple hybridisation fragments of different sizes indicated a highly complex insertion event. the fourth mutant explored as a control in this study was the previously characterised non-virulent f. graminearum strain tp. this strain was previously demonstrated in a targeted pcr based analysis to harbour a single insertion event in the top gene and was generated using the same plasmid.figure dna blot analysis of three mutants showing low, medium and complex insertion events. daf mutant genomic dna was digested with two restriction enzymes and probed with a radioactive labelled hph gene. lanes to 3: ecori digested dna of daf daf and daf lanes to 6: xhoi digested dna of daf daf daf sequencing and findinsertseq bioinformatics approach to identify insertion tags and transformation-induced mutations illumina sequencing was used to obtain high whole-genome coverage for the three daf mutants and the control strain tp. genome coverage of to times was achieved by 100-bp paired-end reads from inserts with a mean library fragment size of 430 bp on an illumina hiseq system. to detect the plasmid insertion sites an analysis pipeline, called findinsertseq, was devised. the 5′- and 3′-mate sequences of the paired-end reads were mapped to the plasmid reference sequence. the pairs where both mates did not map to the plasmid were discarded. the pairs where one mate mapped to the plasmid and the other mate did not map to it, were presumed to be located at the border of the plasmid and the fusarium genomic dna. from these desirable sequences, the latter of the pair was extracted. in these pairs the unmapped mate represented either the forward or reverse sequences that corresponded to either side of the inserted plasmid sequence. these extracted unmapped mate reads were then mapped to the f. graminearum reference genome, already available for the strain ph- resulting in two stacks of reads for each plasmid sequence insertion. for the f. graminearum strain tp with a single plasmid insertion site in chromosome sequence read stacks for the two plasmid flanks are displayed. the identification of loci marked by the two read stacks was achieved by sorting the mapped reads by base pair position in the sequence alignment map file. then a python script using a sliding window of 300 bp to count mapped reads was used to identify where the read coverage peaked. the peak identified represents both the forward and reverse stacks and is reported by the start and stop base pair positions of the 300-bp window and the chromosome number. for mutants of interest, confirmation of the flanking regions reported by the pipeline and any deletion of reference sequence via the gap in coverage between these flanks was verified using a genome browser software such as tablet. this was used in combination with a binary alignment map file generated by mapping the unmapped mates from the plasmid to the ph- reference. however, in genome regions where no reference sequence was available, or where no read stacks were found suggesting that the genomic reference was incomplete another approach was required. the extracted reads were de novo assembled to compile the genomic sequences flanking the inserted plasmid. this later approach was required to resolve the insertions sites of mutants daf and daf.figure findinsertseq: sequencing workflow to identify dna tag insertion sites and corresponding flanking genomic sequences. step paired-end reads are mapped to the plasmid reference sequence, step extract unmapped mate reads from the paired partners that should contain sequences either side of the inserted plasmid sequence. map the extracted mate reads to the f. graminearum reference genome which should form two stacks for each plasmid sequence insertion with one in the forward orientation which are mated with the reads that originally mapped to the plasmid at the 5′-end and a stack with reverse orientated matching reads originally mapped to the plasmid at the 3′ end. step a) the availability of reference genome is a prerequisite for this workflow. b) * if no reference genome is available de novo assembly of reads to identify flanking regions could be considered followed by blast and pcr analysis to characterise the insertion site. step to locate the stack chromosome and bp positions, the mapped reads are sorted, positioned and printed to the command line. additionally the results are printed to an individual sample text file. open tablet software with the mapped genomic reference bam file and proceed to the chromosome and position that were identified in step to visualise the insert flanking sequences and any deletion of reference sequence via the gap in coverage between the two stacks. when a reference genome assembly is either not available or both flanking sequences have not been detected due to the local incompleteness of the reference genome, then de novo assembly of the extracted unmapped mate reads can be used to reconstruct the sequences replacing steps a through with step b. visualisation of a single insertion site in f. graminearum strain tp. sequence reads are visualised in a screenshot using tablet software. sequence reads mapped to this region of the genome, coloured lines represent the genomic sequence of the visualised region and frame translations, sequence coverage histogram, and mapped reads for both flanking regions and the narrow gap between the two read stacks which marks the position of the plasmid insertion. forward green stacks and reverse purple stacks are shown. mapping the insertions findinsertseq localised the insertions in chromosome for strain daf and daf and chromosome for daf and tp. for daf the tag insertion into chromosome was accompanied with a small 12-bp deletion. for tp, our wgs analysis confirmed the result obtained by baldwin et al. that the plasmid insertion into chromosome caused a 4-bp deletion. tag insertion in daf and daf were accompanied with more extensive deletions next to the insertion sites. for daf the insertion occurred downstream of a telomeric sequence repeat.table location of insertion tags and deletion events at the insertion site isolate chromosome location sequence at the 5′ end of the insertion supercontig sequence deletion* sequence at the 3′ end of the insertion plasmid insertion site taaccc † not identified *where the deletion is large, bp positions are given. †telomere sequence taaccc given in italics. for three of the strains the plasmid insert positions were established by mapping to the plasmid and subsequent mapping of mate reads to the reference genome. whereas for daf de novo assembly was necessary to identify the sequence at the 5′ end of the insertion due to an incomplete reference sequence in this part of the f. graminearum genome. the 5′-flanking insertion sequence was not identified for daf using either approach. identification of chromosome deletions and amplifications the sequencing of the mutant genomes permits further qualitative analysis which may be missed using molecular biology methodologies resulting in falsely linking a retrieved gene sequence to a phenotype. for each of the daf mutants, the obtained genomic reads were mapped to the f. graminearum reference genome. this analysis detected additional deletion and/or copy number variations of genomic sequence associated with the plasmid insertion process. other formal possibilities by which variation could have been introduced are cellular stress responses induced by the transformation method, growth and storage conditions. in the tp mutant no detectable amplifications or deletions other than at the plasmid insertion site were detected. for daf the dna gel blot analysis had predicted a single plasmid insertion event into the genomic dna. however, this further sequence analysis revealed that besides the insertion in chromosome an additional untagged chromosomal deletion had occurred. this untagged deletion event had resulted in the loss of  bp at the 3′-end of chromosome predicted to contain genes. for daf the dna gel blot analysis suggested the presence of potentially two copies of the plasmid. the wgs analysis revealed that the single insertion site was accompanied with a -kb deletion close to the 5′-end of chromosome resulting in the loss of genes upstream. however, further inspection of the daf wgs data also identified a twofold amplification of  mb immediately downstream of the deletion event. for daf the dna gel blot analysis had suggested a highly complex insertion event. the plasmid insert in daf resulted in the loss of  kb at the 5′-end of chromosome but had left the telomere intact. the deleted genomic region in daf is predicted to contain genes.figure schematic overview of chromosomal recombination and deletion events for the three daf mutants and verification by diagnostic pcr. daf partial chromosomal deletion and insertion event. for comparison the chromosomal deletion for mutant daf published earlier by baldwin et al. is indicated. pcr analysis of chromosomal regions predicted to have either been lost or retained on chromosome in strain daf the wild type and daf genomic dna was amplified with oligonucleotide pairs in lanes: u516/u u518/ u550/u u549/u u606/u u520/u and u522/u the approximate location of the amplified pcr fragments is indicated in the schematic overview from left to right corresponding to pcr products in lane to daf shows a single-copy insertion in chromosome and a large untagged deletion on chromosome diagnostic pcr in lanes: u533/u u553/u u603/u u535/u approximate location of fragments is indicated in the schematic from left to right. diagnostic pcr of wild type and daf chromosome in lanes: u540/u u541/u and u556/u approximate location of fragments is indicated in the daf schematic from left to right. in daf three multimerised plasmids are inserted at position bp in chromosome diagnostic pcr of wild type and daf chromosome in lanes: u600/u u558/u u545/u u545/u u604/u and u547/u approximate location of fragments is indicated in the schematic from left to right. large black lines indicate the chromosomes. chromosomal deletions, plasmid insertion point. diagnostic pcr fragments. dna ladder bsteii. sequence coverage line graphs of characteristic differences at the genome level for the three daf mutants compared to the control strain ph-1 top-1. average numbers of sequence reads per bp interval were mapped to the wild type reference genome. top panel, daf partial chromosome deletion event. middle panel, daf partial chromosome two deletion event. bottom panel, daf partial chromosome four deletion event and twofold amplification of a mb region downstream of the deletion event. the open triangle in each daf mutant plot indicates the position of the loss in genomic sequence. for each mutant, the corresponding chromosome in the ph- top mutant is depicted as a control. deletion of gene regions in the four mutants and numbers of genes lost strain chromosome position of sequence loss number of genes contained within the lost regions *for daf the plasmid vector insertion occurred in chromosome. chromosome was affected by an untagged gene deletion event. estimation of plasmid copy number for two of the daf mutants, daf and daf the plasmid copy number inferred from the dna gel blot analysis and the number of insertion sites detected within the f. graminearum genome by wgs analysis did not precisely match. therefore an estimation of plasmid copy number by wgs was attempted for all four mutants. to achieve this, the coverage histograms of tp and the three daf mutants were plotted across the whole plasmid sequence of the vector phyg to identify whether there had been recombination of the plasmid resulting in incomplete plasmid sequence insertion. average calculated genome wide coverage for the tp strain was vs. the experimental observed value of for the mapped reads to the plasmid sequence. this suggested an approximate × plasmid length insertion in tp, however only 1× plasmid length insertion was detected from the de novo assembly. this result suggests that this wgs–plasmid alignment approach for the estimation of plasmid copy number is fairly accurate.figure coverage histogram of reads mapped to the inserted plasmid in the four mutants. rows are: tp daf daf and daf the experimentally obtained plasmid average read coverage x¯ for each mutant is given on the far right. the reference genome read coverage for tp is. row depicts the genetic elements of the insertion plasmid. trpc promoter, hygromycin-phosphotransferase, f origin of replication, ampicillin and e. coli oric. coverage of the three daf strains across the plasmid sequence was found to be variable with either some segments absent and/or other segments with increased read density indicating multiple copies of plasmid sequence at the insertion site. in daf the plasmid had an average observed coverage of suggesting an insertion sequence length of approximately plasmids based upon a calculated read-coverage of however some segments of the sequence were over represented. a similar case was found for sample daf with an average coverage of but a calculated coverage of suggesting a length of three plasmids inserted into the genome. pcr amplification of the concatenated plasmid is hindered by the large size of the concatemer and due to the fact that it consists of repeated elements. similarly, de novo assembly is hampered due to the repetitive nature of the concatemer that had formed. the data obtained for daf suggests a single plasmid insertion of 50% of the length of the sequence with an observed coverage of in the regions where coverage was evident vs. a calculated coverage of both tp and daf insertion sequences were validated using de novo assembly and support the reported findings. interestingly, the plasmid gene cassettes noticeably amplified in daf daf and daf include the resistance marker hygromycin-phosphotransferase and the bacterial ampicillin resistance gene while the e. coli oric replication sequences of the plasmid vector were found to be less frequently amplified. collectively, these data indicate that multiple plasmid concatenations at a single insertion site had occurred in both the daf and daf mutants. experimental verification of insertions and chromosomal deletions by pcr to demonstrate that the computationally predicted plasmid insertion sites can be verified in the laboratory, insertions and chromosomal deletions were confirmed using a pcr based approach. this test also excluded the possibility of translocations of affected chromosomal regions into other chromosomes. primers were designed to detect the presence or absence of ~ -kb diagnostic fragments across the modified chromosomes. plasmid insertion sites were verified by designing oligomer pairs where one primer site lies within the inserted plasmid and the corresponding primer site lies within the adjacent chromosomal neighbourhood. for daf four diagnostic fragments located in the region of chromosome predicted to be deleted, could not be amplified while amplification of wild type dna produced the expected fragments. two further diagnostic fragments upstream of deleted chromosomal region were amplified from both daf and wt dna. a diagnostic fragment originating from a primer site located in the inserted plasmid and from the adjacent chromosomal neighbourhood was only amplified in daf but not in wt dna. the results verified the computationally predicted chromosome modifications. for daf a similar approach confirmed the plasmid insertion site in chromosome in this mutant whole-genome analysis had also revealed an untagged chromosomal deletion in chromosome the deletion and its extent were confirmed using three diagnostic fragments. for daf plasmid insertion and a chromosomal deletion was confirmed for chromosome the exact plasmid insertion sites were further confirmed by sequencing the diagnostic fragments obtained using the plasmid specific oligomers. these data independently confirm the insertion and chromosomal deletion results predicted using our whole-genome sequencing and bioinformatics data analysis pipeline described in this study. virulence genes identified close to the insertion points or in chromosomal deletions the adjacent gene close to the plasmid insertion points and genes contained in the chromosomal regions deleted in mutants daf daf and daf were analysed by blast analysis against the pathogen-host interactions database composed of experimentally verified, virulence and effector proteins from bacteria, fungi and oomycete pathogens that infect a wide range of hosts. phi-base associates gene and protein information with published fungal mutant phenotypes. these phenotypes include altered virulence phenotypes, unaffected pathogenicity, and lethal. in the deleted regions of daf daf daf the blast analysis revealed the presence of and phi-base homologs with the phenotype “altered virulence” at e ≤ . we conclude that the absence of these functional homologs of virulence-associated genes is responsible for the pathogenicity defect of the three mutants on wheat. the blast analysis also revealed that in the three mutants a total of five putative transcription factor genes had been deleted that were previously implicated to be essential genes in f. graminearum. in a genome-wide single gene deletion study of transcription factors no successful reverse genetics mutants could be obtained. these genes are in daf fgsg_, in daf fgsg_, fgsg_ and in daf fgsg_, fgsg_. the absence of these transcription factor genes was confirmed by pcr amplification using the wild-type strain ph- as the positive control. we conclude that the five transcription factor genes are not required for growth in the characterised f. graminearum insertion mutants generated in the strain ph- random sampling subsampling of the illumina sequence read dataset was computationally performed for the tp mutant strain using three different seed values. the original data set had an approximate coverage of the seqtk program was used to produce three technical replicates for 10%, 25%, and 50% of the total reads. the average genome coverage for each data set is and respectively. visual inspection of the datasets identifies that the insertion tag can unambiguously be identified in all three replicates for the 25% and 50% subsamples but not for the 10% subsample. we conclude that a 22× coverage obtained by the illumina highseq sequencing platform is sufficient for f. graminearum to identify insertion tags.figure random data sampling to determine the minimal genome coverage required for insertion point characterisation in f. graminearum strain tp11. sampling with three reduced data replicates of 10% each. the deletion sequence between 5′ and 3′ read stacks is represented with an arrow in aa. sampling replicates with 25% of data. sampling replicates with 50% of data. the average coverage for each data set in a, b and c at the insertion region is and respectively. the original data set had an approximate coverage of discussion in this study we setup and describe a bioinformatics workflow called findinsertseq to efficiently characterise forward genetics mutants created by plasmid insertion in the filamentous plant pathogen fusarium graminearum using illumina whole-genome sequencing approach with paired-end reads. the four mutants selected showed a reduced virulence phenotype on their host wheat. the mutants were selected to include low and complex insertion events visualised by dna blot analysis. using the findinsertseq pipeline we were able to rapidly identify dna tag insertion points to the nucleotide level for the control strain tp and three novel disease attenuated fusarium mutants generated for this study. the principle of the wgs-findinsertseq analysis approach is to map sequence reads to an insertion vector and use the mate that aligns to the genomic reads to identify the insertion point in the genome. findinsertseq can also be used to discriminate between single insertion and complex tandem insertion events in a mutant where a reference sequence is available for the species or could be adapted where a reference genome is lacking. thirdly, when de novo mapped read coverage comparisons of mutant and wildtype dna were included in the method, this revealed the existence of further unexpected recombination events, such as chromosomal deletions and amplifications. to our knowledge this study, for the first time, demonstrates the analyses of complex insertion events on the whole-genome level in a pathogenic filamentous fungus of medium 37-mb-genome size. the predictions made using the findinsertseq pipeline were independently confirmed through diagnostic pcr analysis. current laboratory-based methods to determine insertion points such as tail-pcr and plasmid rescue can, in most cases, only successfully be used to identify the insertion event where a single copy of the insertion tag has occurred. many research groups over the past 20 years have created mutant libraries of which only a small number of single-copy insertion mutants were successfully analysed. such insertion mutant collections are now amenable for further analysis using wgs analysis and the findinsertseq pipeline. for sequencing we used the illumina hiseq platform, which is currently the standard high-throughput sequencing method. sequencing of an 800-bp fragment library using 100-bp paired-end reads generated ~100× fold f. graminearum genome coverage when four samples were pooled across one sequencing lane. the size of the fragment library is an important factor. if the fragment size exceeds the insertion vector length, an offset of the insertion point will occur. this offset can be calculated as mean library fragment length minus insertion vector length. the choice of mapping software can influence the success of the workflow. a comparison of novoalign, bowtie and bwa mappers using the default settings showed that only the first two successfully mapped the insert. however, the bwa aligner did not map the reads to the correct position in three of the four strains analysed. this result was attributed to the loss of direction of the paired reads because both stacks of the unmapped mate reads were represented in the forward orientation. an alternative strategy was considered via de novo genome assembly followed by applying blast to identify the insertion site of the plasmid. however such an assembly-based approach was not implemented due to the higher computational resources required to assemble each mutant compared to mapping first to a short plasmid sequence, followed by aligning a small subset of reads to the reference genome. for species with larger genomes i.e. plants, plant-infecting insects or pathogenic protists de novo assembly is challenging and often dependent on many non-intuitive parameters such as k-mer value selection. in addition the output from the mapping approach provides three visually compelling features that the insertion site has been correctly identified. the characteristic features are: two flanks with a similar number of aligned reads separated by a clear gap where the plasmid inserted and deleted a variable number of nucleotides. the findinsertseq workflow currently advises visual inspection using the tablet software as an optional step for mutants of further interest, to inspect forward and reverse sequence stacks either side of the insertion point and identify the deletion and flank sequence to 1 bp. currently only an approximate location of the insertion is given for screening purposes of the first and last base of the highest read coverage peak using a set window size. this location is written to output but surmises the region of the genome in which the insertion took place. this approach permits the biologist to experimentally confirm the insertion point using a pcr-based analysis. randomly down-sizing the sampled genomic read coverage for the control mutant strain tp revealed that only x genomic coverage is required to identify the insertion site with a cost incurred per sample of approximately $ including all associated cost such as dna preparation and library construction. sequencing methods are evolving rapidly and as a consequence associated costs are expected to drop even further. many fungal genomes are of similar size to f. graminearum and have a similar gc content. a wgs-findinsertseq approach can identify multiple locations of plasmid insertions. however, this may prove more difficult to apply in fungal eukaryotes such as magnaporthe oryzae for which % repetitive dna is reported. if the insertion event flanking sequence contains repeat sequences, mapping will be ambiguous. transformation events may result in reshuffling and partial amplification of the insertion tag. we found this strikingly to be the case for the mutant strain daf the coverage histogram revealed that the resistance marker gene encoding hygromycin-phosphotransferase was highly amplified during vector plasmid integration as well as the ampicillin gene cassette. these events could be favoured by dna structural elements in the vector influencing the outcome of recombination events. the amplification of the hph gene is likely to be selected for when growing the transformants on hygromycin-containing medium. in addition to the pipeline described in figure  we investigated situations where only a single flanking sequence was identified. this outcome suggested further genomic alterations might have occurred. in these cases all genomic reads from the mutant were mapped back to the reference. this approach successfully revealed additional tagged and untagged chromosomal deletions and amplifications events in the mutants. the extent of the chromosomal deletions is surprising and suggests that larger parts of the f. graminearum genome are not required for growth in vitro and / or during the protoplast transformation and regeneration procedure used in this study. similar results were found for two f. graminearum mutants, called z43r and daf where the occurrence of large chromosomal deletion events was reported earlier. in both studies a combination of plasmid-rescue and sanger single-read sequencing was used to characterise the mutants. in z43r a genomic region of 220 kb containing at least genes was deleted, whilst in daf a genomic region of 350 kb containing genes was deleted. the molecular analysis of z43r revealed a deletion of a genomic region at the vector insertion site, including the gene clusters required for the biosynthesis of aurofusarin and zearalenone. the authors deliberately recreated the deletion event using a reverse genetics approach to demonstrate that a large region of genomic dna can be efficiently deleted in f. graminearum by double homologous recombination. in the daf study, the unintentional deletion of a large region of genomic dna permitted the authors to report that the loss of the tri gene involved in mycotoxin biosynthesis was responsible for the virulence defect on its host. interestingly, the mutant daf recreated the daf mutant and extended the deleted region by an additional genes. taken together, these findings show that large chromosomal regions can systematically be deleted in f. graminearum to understand genes sets and genome organisation. in s. cerevisiae, such an approach is currently being used to build a minimalist yeast genome within the sc project. the deleted chromosomal regions in the analysed mutants identified a total of genes that when deleted from the f. graminearum genome are not required for life and are thus unlikely to make good fungicide targets. within this gene set were five putative transcription factors that were earlier proposed to be essential for life in f. graminearum. son and colleagues tested in a genome-wide gene deletion study putative transcription factors, but no gene deletion mutants could be obtained for these five genes. while it is possible that the absence of these proteins is compensated by the absence of additional proteins within the deleted chromosomal regions, further experiments are needed to elucidate their function.  this study demonstrates that whole-genome sequence analysis is an efficient tool to characterise forward genetics mutants in the filamentous pathogenic species f. graminearum. surprisingly in some cases the dna tag insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. this fact suggests that the findinsertseq method identifies dna tag insertion points and other genomic recombination events such as chromosomal deletions and dna amplifications. large scale chromosomal deletions can quickly identify the non-essential gene set or host-specificity genes not essential for pathogenic life. the minimal sequencing coverage required in f. graminearum to survey accurately the genome to a single base level for recombination events was determined to be 22× fold. findinsertseq will also allow full characterisation of recombinant filamentous fungal isolates overexpressing heterologous proteins for research and/or industrial scale commercial applications. 
this study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete fusarium graminearum. a random data sampling approach revealed the minimum genome coverage required to survey the f. graminearum genome for alterations. the workflow was tested using four mutants showing evidence of single and multi-copy insertions in dna blot analysis. plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. in some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. we developed the findinsertseq workflow to localise the dna tag insertions to the nucleotide level. we used a whole-genome sequencing approach using illumina hiseq technology to investigate dna tag insertion points and chromosomal deletion events in mutagenised, reduced virulence f. graminearum isolates identified in disease tests on wheat. by comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and dna amplification were observed in three of the analysed mutants.
 identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.  we used a whole-genome sequencing approach using illumina hiseq technology to investigate dna tag insertion points and chromosomal deletion events in mutagenised, reduced virulence f. graminearum isolates identified in disease tests on wheat. we developed the findinsertseq workflow to localise the dna tag insertions to the nucleotide level. the workflow was tested using four mutants showing evidence of single and multi-copy insertions in dna blot analysis. findinsertseq was able to identify both single and multi-copy concatenation insertion sites. by comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and dna amplification were observed in three of the analysed mutants. a random data sampling approach revealed the minimum genome coverage required to survey the f. graminearum genome for alterations.  this study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete fusarium graminearum. in some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. the findinsertseq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords virulence mutant analysisgibberella zeaeforward geneticsnext generation sequencingfilamentous plant pathogenissue-copyright-statement© the author 2015 