BACKGROUND
the national public health laboratory tel aviv, national mycobacterium reference center routinely genotypes all new culture-positive tuberculosis cases in israel  <cit>  with  <dig> spacers reverse line blot spacer oligonucleotide typing  , and  <dig> loci mycobacterial interspersed repeat units – variable number tandem repeats  typing  <cit>  using multiplex pcr and capillary electrophoresis  <cit> .
the results are stored in excel , bionumerics , and whonet databases.
from 2008– <dig>  over  <dig> strains were genotyped and tested for drug sensitivity to first line, and when necessary, second line drugs.
six drug-sensitive isolates from three different patients a, b, and c  yielded negative spoligotyping results and three different  <dig> loci miru-vntr profiles respectively .
variation in the structure of crispr and crispr-flanking regions of these negative spoligotype mtbc isolates was observed when ngs reads were mapped on reference whole genome sequence h37rv nc_ <dig> .
this yielded visualization of coverage .
after substitution of manual-pcr-derived dr-spanning sequences for the corresponding map-derived regions, further details of structural variation were revealed by alignments of annotated subsequences containing crispr plus crispr-flanking regions to the annotated reference h37rv sequence .fig  <dig> location and coverage of h37rv-mapped ngs-reads of negative-spoligotype strains a, b, c and intact-dr t3_eth
fig  <dig> structure and variation of crispr and crispr-flanking regions in deleted-dr and intact-dr mtbc
in addition to the  <dig> drug-sensitive deleted-dr isolates, two drug sensitive and  <dig> multiple-drug-resistant  intact-dr strains from t3_eth , our largest african-origin mdr-containing cluster, were analyzed by ngs .
these provided positive controls for the sensitivity of our methods to correctly characterize from ngs data, the crispr-cas regions of intact-dr mtbc, especially with respect to the presence or absence of crispr spacers.table  <dig> characteristics of ngs sequenced deleted-dr sample strains a, b, c and intact-dr t3_eth, and whole genome sequences obtained on-line or assembled from on-line read sets#
mutt4
t2
ogt
#id, sample, read set or ncbi accession number; octal, from spoligosome <dig>  sit, spoligotype international type and associated family; ns, no sit; drug res, resistant to drug, i-isoniazide, r-rifampicin, e- ethanbutol, s-streptomycin, p-pyrazinamide, ds : none, dr : at least one but not i&r together: mdr : at least i&r, xdr : at least mdr plus defined 2nd line drugs consisting of at least  <dig> quinolone and at least one of the injectable drugs kanamycin, capreomycin or amikacin, tdr : resistant to all 1st and 2nd line drugs.
a-g: additional file 2: list s <dig> – sources of drug sensitivity data; wt, identical to reference genome h37rv nc_ <dig> ; crispr is <dig>  orientation of the is <dig> mobile element found in the direct repeat region, wt: same orientation as in h37rv, inv: inverted, unk: unknown; mutation in mutt <dig> , codon 48: arg cgg ggg gly; mutation in mutt <dig> , codon 58: gly gga cga arg; mutation in ogt , silent codon 12: gly ccc tcc gly, missense codon 15: thr ggt gct ser ; regions of difference rd, 105: proto and all modern beijing, 207: lsp in crispr region defining modern beijing – includes cas gene deletions, 181-150-142: polymorphic lsps for beijing, 182: marker for haarlem, 239: marker for eai, 750: marker for cas
complete loss of all direct repeat region  repeats and spacers, negative spoligotyping results, and loss of genes in flanking regions has been reported previously for mtbc with negative spoligotypes  <cit>  identical to those currently assigned to sit  <dig>  <cit>  and for other bacteria  <cit> , however, the molecular structures of the crispr regions  and flanking crispr-associated  regions  <cit>  were not reported, except for a comparison to the homologous genomic region in non-tuberculous mycobacteria   <cit> .
the evidence reported herein clearly showed that the deleted-dr and accompanying deleted crispr-flanking genes of the mtbc described in this study were not an instance of the orfs absent in ntm species .
also, this study is the first known to us to employ identification of region of difference  deletions in order to assign negative spoligotype mtbc to their corresponding major spoligotype families.
the mtbc rd deletions are genomic long sequence polymorphisms   <cit>  which were detected in this study by aligning mycobacterium tuberculosis complete genome sequences  with the mtbc reference strain h37rv complete genome sequence  <cit> .
the mtbc rd deletions, and also an extensive set of single nucleotide polymorphisms , have been shown by coll et al.
<cit>  in an on-line available application for analysis of ngs reads, to have phylogenetic behavior that corresponds well with the phylogenetic distribution obtained by analysis of 43-spacer spoligotypes.
43-spacer spoligotypes have been assigned type numbers and family names  <cit> .
the 43-spacer spoligotypes are more informative than the rd types and snp types, thus any given specific 43-spacer spoligotype cannot be inferred from an rd type or snp type, but the spoligotype family can be inferred from rd or snp information, and in addition some sub-lineage information can be obtained from the snp type  <cit> .
therefore, when one encounters a negative spoligotype by conventional screening for the  <dig> crispr spacers, if ngs sequencing can be performed for the deleted-dr strain, two convenient ways exist to determine the spoligotype family: 1) map the ngs reads to reference h37rv, align the constructed mtbc sequence with the h37rv sequence, and search for the relevant rd deletions, and 2) submit the ngs reads to the coll et al.
<cit>  site for analysis of rd deletions and snps.
the rd data shown in table  <dig> suggested that a third way could be developed which would not require sequencing for ngs reads: a possible multiplex pcr probe for relevant rd deletions, which could be analyzed by fragment analysis as in miru-vntr typing, or by real time pcr.
various methods have been proposed for spoligotyping from ngs reads , but all involve detection of crispr spacers and thus would only yield negative results with our deleted-dr negative spoligotype strains.
a combination of the pcr-based methods  <dig> spacer spoligotyping and  <dig> loci miru-vntr typing has replaced rflp-typing as the mtbc genotyping screen of choice, due to the speed and convenience of the pcr-based methods, genotype discrimination as good as or better than that for rflp-typing, and the ability to store the pcr-based results in easily manipulated digital format  <cit> .
on a population level, screening for spoligotype family provides insight into the bio-geographic burden of tb in the world and for any particular country  <cit> .
thus it is useful to be able to determine the spoligotype family for a strain that has a negative spoligotype by conventional screening.
while in general the more informative  <dig> loci miru-vntr type will still phylogenetic-wise segregate according to its complementary spoligotype  <cit> , for certain miru-vntr profiles the miru-spoligo correlation may be difficult to establish.
a published example of this  <cit>  showed that the miru-vntr profiles in spoligotype families cas and beijing can sometimes be difficult to distinguish.
in another case, very similar miru-vntr profiles were found in both the t and lam spoligotype families .
in these cases it was not only useful, but indeed necessary, to determine at least the spoligotype family.
on a clinical level, the combination of spoligotype and miru-vntr type complements and guides classical epidemiological investigation to clarify questions of transmission between individuals, transmission chains among larger groups of individuals, questions about whether a recurrence of tb is due to reactivation or reinfection, and resolution of questions arising when a lab provides a positive tb report that is inconsistent with the physician's observation – that is- the possibility of a false positive due to lab contamination.
often  <dig> loci miru-vntr alone supplementing classical epidemiology is enough to resolve these issues.
nevertheless, if the strains involved have negative spoligotypes on conventional screening, knowledge of at least the spoligotype family  can help validate the results concluded from classical epidemiology supplemented with miru-vntr typing.
finally, there are often gaps in epidemiological knowledge when trying to establish transmission chains among larger groups of individuals.
these knowledge gaps can be exacerbated by subtle polymorphisms  in miru-vntr profile, where knowing only the spoligotype family will not be enough to firmly establish the transmission chain.
therefore, when confronted with negative spoligotypes in large putative transmission chains, it seems reasonable to recommend that all the strains should be processed for ngs reads.
then the ngs reads for the negative spoligotype strains should be used for spoligotype family determination  <cit> , and subsequently for snp clonality-check comparisons among the strains to delineate the outbreak  <cit> .
mycobacterium tuberculosis complex  bacteria possess a type iii-a crispr-cas system structurally homologous to type iii-a of other genera  <cit> .
active type iii-a systems provide adaptive immunity to bacteriophage and plasmids  <cit> , and perform endogenous roles, for example, endogenous dna repair via cas <dig> endonuclease  <cit> .
in mtbc the crispr region is also known as the direct repeat region .
polymorphism in the number and type of  <dig> spacers in the dr/crispr region of any given mtbc strain, has been and continues to be extensively used for molecular epidemiology of mtbc, especially for overviews of phylogenetic and bio-geographical distributions of clades  <cit> .
fourty three-spacer-spo-plus-24-loci-miru genotyping provides molecular epidemiology at the resolution of clonal transmission  <cit> , with the exception of certain instances, for example, of convergent evolution  <cit> .
the functional activity of the mtbc crispr-cas system remains to be investigated, but the highly conserved sequence and order of the  <dig> crispr/dr region spacers  <cit>  which is the basis for the epidemiological and phylogenetic usefulness of polymorphism on spacer oligonucleotide typing , has been interpreted to imply at least partial inactivity of the mtbc crispr-cas system  <cit> .
this interpretation presumes that the insertion of new spacers in the crispr/dr region is one of the fundamentals of, and evidence for, adaptive immunity, and thus lack of evidence for insertion of new spacers in mtbc implies at least partial inactivation of the crispr-cas adaptive immune system in mtbc.
the mtbc type iii-a crispr-cas system  <cit>  is defined by the presence of cas10/csm <dig> and csm accessory genes, and is predicted to be in some manner active on the basis of the presence of intact cas <dig> and cas <dig> genes.
in addition, some sort of activity even if only in endogenous roles, can be predicted from genbank annotation that informs of mass spectroscopy support  <cit>  for the existence of most of the cas gene products, including cas1in the reference mtbc strain h37rv.
the lineage-defining rd  <dig> deletion  <cit>  of the super-spreader mdr-containing beijing family includes complete deletion of cas <dig> and cas <dig> genes , so possession of a fully active type iii-a crispr-cas system is not a prerequisite for a successful  mtbc lineage.
interestingly, the complete loss of crispr spacers in e. coli gave the same dna-repair deficient phenotype as deletion of the e. coli cas <dig>  gene  <cit> .
using m. smegmatis as a model for mtbc  <cit> , cas <dig>  <cit>  by itself was shown to be deleterious to mycobacterium survival, thus it may be significant that deletion of cas <dig> was always accompanied by deletion of at least cas <dig>
in summary, the results showed that complete deletion of dr region repeats and spacers in negative-spoligotype mtbc was accompanied by clonal-specific loss of flanking genes, always accompanied by complete deletion of cas <dig>  complete deletion of putative  dna-repair gene cas <dig>  and retention of the middle is <dig> mobile element.
for the first time it was shown that negative spoligotype mtbc nevertheless could be assigned to major spoligotype families by identification of ngs-revealed corresponding lineage-defining rd deletions.
since there was only  <dig> deleted-dr strain-type  per rd-defined family, there was not enough information to decide if the loss of cas genes was solely clonally specific, or perhaps had lineage-defining features.
methods
growth of mycobacterium tuberculosis strains, and drug sensitivity tests
all approximately  <dig> new culture-positive mtbc strains collected during 2008– <dig> were analyzed.
cultures were grown and identified as mtbc as previously described  <cit> .
drug sensitivity tests were performed as previously described  <cit> .
extraction of mtbc dna
preparations of rflp-grade mtbc dna for ngs sequencing, and of “crude” sonicated, heated extract for genotyping, were done as previously described  <cit> .
genotyping of mtbc dna
routine genotyping of mtbc dna by  <dig> spacer spoligotyping  and  <dig> loci miru-vntr typing  were done as previously described  <cit> .
bcgp <dig> was the generous gift of professor hillel bercovier, hebrew university of jerusalem.
european communicable disease center proficiency test  samples  <dig> and  <dig>  and ecdcpt h37rv control dna were obtained originally as proficiency test materials  <cit> .
genotyping results were stored in an excel  database, a bionumerics ver <dig>  database, and a whonet database.
ngs assay and bioinformatics analyses at the scripps research institute, including 1) coverage-curves illustrating deletion of crispr/dr-cas regions present in the template reference h37rv chromosome sequence, and 2) determination of spoligotypes from ngs reads
rflp-grade dna samples  were sent to mgr.
steven robert head and mgr.
lana schaffer of the scripps research institute in san diego, california usa for ngs and draft bioinformatics analysis.
one microgram of each sample was sheared on the covaris s <dig>  each sample was then end repaired, a-tailed and adapter ligated.
the samples were cleaned with agencourt beads and treated with 6 cycles of pcr.
after pcr the samples were cleaned using zymo  <dig> columns and loaded on a 2% agarose gel.
the gel was cut from 400–700 bp and cleaned again with the zymo <dig> column.
the samples were sequenced on an illumina miseq system as paired-end reads each with length 300 bp.
the alignment was performed using novoalign v <dig> .
<dig>  without soft clipping to allow reads which cross the ends of the reference sequence as would be the case of a circular genome.
the mycobacterium tuberculosis reference sequence used for the alignment was mtb-h37rv-asm19595v <dig> and for repeat sequences not in the mapped-to-h37rv mtbc sequence was mycobacterium bovis af2122/ <dig>  the reads were preprocessed by trimming adaptor sequences , trimming by base call quality scores lower than  <dig>  removing unmapped reads and duplicate reads, and removal of reads with mapping quality score less than one.
repeat sequence coordinates and sequences were taken from the jansen paper  <cit> .
for coverage curves illustrating deletion of crispr-dr-cas regions that were present in the template h37rv chromosome sequence, the aligned bam files were converted to wig files using bamtools, bedtools, and ucsc-tools.
the wig files were imported into ucsc microbial genome viewer .
recovery of spoligotypes from ngs data was accomplished as follows.
a bed file was created using the coordinates of the  <dig> dr repeat units.
this bedfile and the aligned bam files were used with “samtools bedcov” tool to output number of counts for each repeat unit.
in order to produce a binary table matching the spoligotyping regions with less than  <dig> counts were considered absent.
bioinformatics at the national public health laboratory tel aviv
analyses of our ngs raw data were accomplished by use of bionumerics version  <dig>  and  <dig>   import and processing of 1) cleaned paired-end and single-end fastq reads received from scripps, 2) reads formatted as sra  and fastq files downloaded from ncbi or other sources, and 3) fastq or embo/gb format reference whole chromosome sequences or contributed chromosome sequences from ncbi or other sources.
for visualization of the molecular details of the crispr-dr and flanking regions , reads were resequenced/mapped onto reference h37rv nc_ <dig>  using default parameters with the following changes: accept paired-end reads, minimum coverage  <dig>  minimum sequence length before considered for contig 500 nt.
the “coverage” was examined as percentage of the h37rv genome matched, and as the number of reads contributing to each base pair assignment.
the mapped chromosomes were annotated with h37rv genbank and bionumerics-predicted  features and matrix compared and aligned in chromosome comparison mode using default parameters.
for decisions about reading frames and codon assignments, reference h37rv nc_ <dig>  annotated only with genbank annotation was included in comparisons.
default parameters were: i. comparison project type: full sequence based; matrix: matrix; self-comparison of sequences; direct sequence orientation; inverted sequence orientation; seed extension dna seed: minimum  <dig> matches in window size of  <dig> bases; amino acid seed: minimum  <dig> matches in window size of  <dig> amino acids; stretch extension minimum  <dig> matches in window size of  <dig> bases; minimal stretch length: 50; ii.
seeds seed database: seed  <dig>  type: dna, length:5; seeds used in project: seed  <dig>  type: transl, length: <dig>  and iii.
alignment settings template entry: usually the gb plus bn annotated reference h37rv; stretch import minimum length: 0; minimum identity: 0; stretch mapping open gap penalty: 10; extend gap penalty 10; do not allow repetition of mapped stretches ; full alignment of stretch ends: checked; full alignment settings: open gap penalty : 100; extend gap penalty : 0; use fast algoritm: checked; k-tuple size:2; no.
of diagonals :  <dig>  this generated a schematic suitable for visualization of deletions, while choosing “show text view” while in alignment sub-mode in the comparison mode allowed visualization of single nucleotide polymorphisms .
an important point is that any given set of parameters has its own strengths and limitations.
the default parameters of bionumerics  <dig>  and  <dig>  chromosome comparison did not detect inverse is <dig> in the crispr-cas region , nor were they suitable for comparison of shorter sequences .
the region containing the crispr-cas sequence plus flanking regions was cut out , annotated and used for further comparisons in the alignment window when high similarity was expected, or in the chromosome comparison mode and alignment sub-mode with appropriate parameters when some dissimilarity was expected.
the appropriate parameters for comparison of shorter sequences in the chromosome comparison mode were : i. comparison seed extension: dna seed: minimum  <dig> matches in window size of  <dig> bases; amino acid seed: minimum two matches in window size of  <dig> amino acids; stretch extension: minimum  <dig> matches in window size of  <dig> bases; minimal stretch length: 12; ii.
seeds seed database: seed: 111; type: dna; length: 3; seeds used in project: seed: 111; type: dna; length: 3; iii.
alignment settings: full alignment settings: open gap penalty : 0; no.
of diagonals :  <dig>  other parameter sets can be better for other uses, and must be determined empirically.
in order to obtain contig-corrected sequences for the six approximately  <dig> kbp crispr plus crispr-flanking subsequences of the deleted-crispr strains from patients a, b, and c, the ngs-obtained h37rv-mapped subsequences containing the crispr-spacers regions  were excised from the assembled mapped whole genome sequence at predetermined flanking genes on each side, and replaced with sequences of pcr validated contigs spanning the crispr-spacers-dr region, that is, with 5’ and 3’ ends of the contigs overlapping the conserved crispr-flanking genes  characteristic for each deleted-dr strain.
these “drplus” sequences corrected for the appropriate contigs are found in additional file 3: sequences s1–s <dig>  the crispr-flanking genes and their locations are listed in additional file 4: table s <dig>
the pcr validated contigs were constructed using primers inferred from the ngs data from mapped to h37rv, and de novo, sequences  according to which regions could be expected to be “conserved” in the crispr-spacers region itself , and in the immediately adjacent crispr-flanking regions characteristic for each deleted-dr strain.
pcr and agarose gel electrophoresis were performed as previously described  <cit>  and the pcr product purified on qiaquick  columns.
the purified pcr product was sent for forward and reverse sequencing to a commercial sequencing facility , and the sequences from the ab <dig> sequencing files were trimmed and used to construct contigs spanning the crispr-spacers region .
validation of sequence trimming and alignment for contig construction was done in parallel using a different bioinformatics tool .
the h37rv nc_ <dig>  whole genome sequence locations, corresponding to the dr plus flanking regions  of the mapped deleted-dr strains a, b, and c, were used for alignments for primer construction .
the major primers used for manual pcr were as shown in additional file 5: table s <dig>
recovery of spoligotypes from ngs reads, or from downloaded reference or contributed chromosomes, was accomplished by constructing a new bionumerics version  <dig>   application, manuscript in preparation.
in addition, the sequences for the dr region-spanning contigs for deleted-dr strains a, b, and c were examined by ncbi blast for sequences corresponding to spacer sequences.
drug sensitivity and lineage information for downloaded reads was obtained either from the literature   <cit>  or by uploading the reads to tb profiler  <cit>  which reported back the drug sensitivities for first and second line drugs, and projected lineages.
rd deletion confirmation of lineages  was obtained from bionumerics chromosome comparison alignments of complete genome sequences  to the reference h37rv sequence.
RESULTS
variation in the crispr and crispr-flanking regions: clonal specific deletion of crispr-flanking regions in deleted-dr strains – including deletion of at least cas <dig> and cas <dig>  and retention of the is <dig> mobile element in deleted-dr strains
in fig.
<dig>  coverage of the homologous reference h37rv sequences was approximately  <dig> reads for each isolate, with the negative spoligotype isolates a, b, and c showing wide gaps to the left and right of the middle sequence.
the middle read-coverage length was suggestive of that of an is <dig> mobile element.
the coverage of the template h37rv genome   ranged from  <dig>  to  <dig> % for the deleted-crispr-spacers-cas strains a, b, and c, and  <dig>  to  <dig> % for the t3_eth strains.
variations in the crispr and crispr-flanking regions of negative spoligotype  mtbc isolates from patients a, b, and c were observed in the visualization of ngs reads mapped on reference h37rv nc_ <dig>   and annotated alignments of drplus  subsequences to the drplus reference h37rv subsequence .
for comparison, intact-dr results from three ngs-sequenced t3_eth were shown.
drplus subsequences from isolates a, b, c, and isolate srr <dig> which was assembled from downloaded reads, were aligned to reference template h37rv subsequence .
a, b, and c – specific  deletions of crispr-flanking genes were observed, including deletions of cas genes to the right of the retained mid is <dig>  all  <dig> deleted-dr strains, retained their mid is <dig> mobile element, and deleted at least cas <dig> and cas <dig> .
the deleted-dr b isolates showed a right-side deletion of the same size and location as that shown in beijing-family strains .
subsequent rd deletion identification of deleted-dr b isolates as beijing , suggested that the b deletion may be an extension of the beijing lineage-defining rd <dig> deletion.
this was further suggested by the fact that the deleted-dr b isolates shared the beijing-specific mutator phenotype snps mutt4|t2|ogt  <cit>  .
thus we propose to name the deletion seen in the deleted-dr b isolates as “extended rd207” in accordance with the terminology adopted by luo et al.
<cit>  for the rd <dig> deletion in proto-beijing: extended rd <dig> .
it was interesting, but of as yet unproven important significance as discussed below, that in contrast to the inverse orientation of the is <dig> mobile element in the dr region of many if not all validated beijing-family sequences  the orientation of is <dig> in the beijing deleted-dr b isolates was wild type, that is, the same as for reference template h37rv .
validation of retention of mid-is <dig> mobile element, and of loss of varying amounts of genomic regions flanking the deleted crispr/dr regions of isolates a, b, and c
in order to validate retention of is <dig>  and clonally specific loss of flanking genomic sequences in deleted-dr isolates a, b, and c, primers were chosen from conserved flanking genes and the mid is <dig>  and used to generate amplicons by manual pcr which were assembled into contigs for a dr-spanning sequence for each isolate a, b, and c. these validated dr-spanning sequences were substituted into the drplus sequences of h37rv-mapped isolates a, b, and c, resulting in a “corrected’ drplus sequence for each isolate a, b, and c .
alignment of the corrected drplus sequences to template h37rv  confirmed that mid-is <dig> was retained in each deleted-dr isolate a, b, and c. it was interesting that the orientation of this is <dig> of each beijing deleted-dr isolate b was wild type with respect to h37rv, and inverse with respect to the orientation of is <dig> observed in reference whole genome beijing sequences .
alignment of the manual pcr corrected dr plus flanking sequences to template h37rv  also confirmed the clonally specific loss of crispr-flanking regions, as shown in fig.
<dig> and detailed in additional file 4: table s <dig>  each deleted-dr isolate a, b, and c showed deletion of at least cas <dig> and cas <dig> from among the cas genes .table  <dig> the presence or absence of mutations in crispr-associated genes in mycobacterium tuberculosis complex strains compared to reference template h37rv nc_ <dig>  4411532 bp.
the mutations are listed in ascending order of their location relative to the reference h37rv chromosome
cas2
cas1
cas1
cas1
cas1
csm6
csm6
csm6
csm6
csm6
csm6
csm5
csm5
csm5
csm4
csm4
csm4
csm4
nonea
csm3
nonea
csm2
cas10/csm1
cas10/csm1
cas10/csm1
cas10/csm1
cas10/csm1
nonea
cas6
anone: no mutations listed for the strains examined; location: base pair of h37rv nc_ <dig>  where snp can be found, or base pair approximately in the center of a given deletion or insertion; ssp short sequence polymorphism, snp single nucleotide polymorphism
the absence of mutations in csm <dig>  csm <dig>  and cas <dig>  could mean that those genes cannot tolerate mutations due to the importance of their sequence, or transcription or translation products.
however, mutations could appear if a larger set of strains were examined, a possibility suggested by the recent report that the cas <dig> gene is dispensable in type iii crispr-cas systems  <cit> .
did the retained is <dig> mobile element have a role in generating the crispr and flanking-sequences deletions observed in the deleted-dr strains?
the mobile element is <dig> inserts into the mycobacterium tuberculosis genome in a variety of ways with a variety of potential effects  <cit> , some of which we have discussed previously  <cit> .
the dr/crispr region is a hot spot for is <dig> insertions, where there can be  <dig>   <dig>  or more is <dig> elements inserted in the dr region  <cit> .
in this study all the mtbc strains examined had  <dig> mid dr is <dig> mobile element .
after crispr deletion, along with deletion of varying amounts of dr-flanking sequences, the dr region was left with a residual is <dig> mobile element attached to what remained of the flanking sequences .
the is <dig> mobile element has the ability to delete sequences on either side of it  <cit> .
in the mtbc beijing family, the mid dr is <dig> mobile element is found in reverse orientation to the h37rv mid dr is <dig> sequence  <cit> .
the results shown in this paper were consistent with these previous findings, as except for the deleted-dr beijing strains from patient b, all beijing mid dr is <dig> elements from validated complete chromosome sequences, had orientations inverse  to the mid dr h37rv is <dig> orientation.
in the deleted-dr beijing strains from patient b, the residual mid dr is <dig> was oriented in the same direction as the reference h37rv mid dr is <dig> mobile element .
we speculate that generation of the deleted-dr extended rd <dig> deletion involved this is <dig> mobile element, which in the process was “flipped” from its previous characteristic beijing inverse orientation  back to a wild type h37rv-like orientation.
the mechanism behind the is <dig> ability to generate deletions has been investigated  <cit> , but to the best of our knowledge this issue has not been resolved.
in summary, it is probable that the residual is <dig> mobile element found attached to the presumed  dr flanking sequences of the deleted-dr strains , played an as yet uncharacterized role in the deletion of the crispr sequences and flanking genes observed to be deleted in the deleted-dr strains .
validation of total loss of crispr spacers in deleted-dr isolates a, b, and c, and in downloaded read-assembled deleted-dr strain srr1188186
approach 1
recovery of spoligotypes from ngs data for isolates a, b, and c, and t3_eth, was accomplished as follows .
a bed file was created using the coordinates of the  <dig> dr repeat units.
this bedfile and the aligned bam files were used with “samtools bedcov” tool to output number of counts for each repeat unit.
in order to produce a binary table matching the spoligotyping, regions with less than  <dig> counts were considered absent.
by this method, the correct spoligotypes  could be obtained for the intact-dr t3_eth.
approach 2
in order to validate the total loss of spacers and repeats in the deleted-dr isolates, for each isolate a, b and c corrected drplus sequences  were examined for spacer and repeat sequences, which were found to be totally absent from these corrected drplus sequences.
approach 3
in order to further confirm the total loss of crispr spacers in deleted-dr isolates a, b, and c, and in downloaded read-assembled deleted-dr strain srr <dig>  a new application was developed  for bionumerics  <dig>  and  <dig>  and excel .
the application yielded results consistent with other approaches  in that no spacers were detected in deleted-dr strains, and correct spoligotypes were assigned to reference h37rv, reference bcg, and the  <dig> sequenced t3_eth.
crispr-cas defects: comparison of mtbc strains for insertions/deletions and snps in cas genes
since isolates from patient b were beijing, it was of interest that the deleted-dr deletion consisted of a left-side-of-is <dig> extension  of the beijing-defining rd <dig> deletion.
the rd <dig> deletion is found in all modern beijing  <cit> , and removes most crispr-spacers and cas genes through rv <dig>    <cit> .
table  <dig> lists the cas gene mutations in order of ascending location on the h37rv chromosome , and the corresponding mutated genes for the strains examined in this study .
notable mutations were the extensive deletions of cas genes in beijing family members including deletion of cas <dig> ; the deletion of cas <dig> and cas <dig> by all deleted-dr strains ; t3_eth specific missense snps in cas1; and a proto-beijing specific missense snp in cas <dig> .
t3_eth is our largest mdr-containing african-origin cluster, and has been reported by others to have proclivity for mdr  <cit> .
how likely is it that the cas <dig> proteins from type iii-a systems in mtbc also have a role in dna repair in a manner similar to that of cas <dig> proteins from type i-e in e. coli?
babu et al.
<dig>  <cit>  induced dna damage in e. coli by exposure to mitomycin c, uv light or cisplatin.
they noted, and gave examples, that cas <dig> proteins from different genera of bacteria can display related but different biochemical properties.
thus any given cas <dig> protein must be characterized for its biochemical and functional roles, which they did for the e. coli type i-e cas <dig>  but which has not yet been done for the mtbc type iii-a cas <dig>  moreover, we and others  <cit>  were not able to find any studies done in any genus to determine the biochemical and functional roles of type iii systems.
nevertheless, makarova et al.
<dig>  <cit>  noted that the cas <dig> family is the most conserved cas protein family  <cit>  and showed that the cas <dig> phylogenetic tree corresponds nicely with the phylogeny of the bacterial genera.
by use of the ncbi tool cdart  <cit> , we observed that the e. coli type i-e cas <dig> had homologous domain architecture with the m. tuberculosis type iii-a cas <dig>  the cas <dig> proteins from both were members of the cas <dig> endonuclease super-family cas1_i-ii-iii.
only the e. coli cas <dig> was labeled “multifunctional” because there is no relevant data yet for m. tuberculosis cas <dig>  however, we consider it likely that the mtbc cas <dig> will eventually be shown to have a role in endogenous dna repair.
it was noted by makarova et al.,  <dig>   <cit>  that the adaptation module  is dispensable in subtypes iii-a and iii-b.
this would tend to support the idea that cas <dig> in mtbc is nonessential, even if it does have an endogenous dna-repair activity role.
consistent with this, the beijing family of mtbc has deleted cas <dig> and cas <dig> , yet still is a very successful pathogen.
however, nonessential does not necessarily mean non-important.
cas <dig> is produced in mtbc  <cit> , except for the possible exceptions noted in our paper , and its absence or mutation-induced defectiveness could in theory result in impaired dna repair and consequent increased mutability.
has induction of the cas <dig> gene been observed in response to dna-damage in genera other than that of e. coli which shows type i-e cas <dig> induction?
pyrococcus furiosus has  <dig> different crispr loci and  <dig> different crispr-cas systems, type iii-b, type i-g, and type i-a  <cit> .
williams et al.,  <dig>   <cit>  analyzed the transcriptome of p. furiosus after induction of dna damage by gamma irradiation.
they observed  <dig> genes that had up to a 10-fold increase in mrna levels following gamma irradiation.
<dig> of the  <dig> genes were crispr-associated, but none of them was cas <dig>  although  <dig> was predicted to be involved in dna repair and recombination  <cit> .
interestingly, all  <dig> up-regulated crispr-associated genes belonged to the type i-a system  <cit> , and none of the genes in the other  <dig> systems, type iii-b and type i-g, were similarly up-regulated  <cit> .
williams et al.,  <dig>   <cit>  note that some efficient dna repair pathways appear to be constitutively expressed in the hyperthermophilic archaeon p. furiosus, which they attribute to the result of life at high temperature.
to our knowledge there are no studies of induction of cas <dig> in response to dna-damage, other than the report of induction of cas <dig> in e. coli  <cit> .
life in a macrophage, and frequent exposure to hypoxic conditions, also lead to a stressful environment for m. tuberculosis, and we ask if this is the reason that cas <dig> was found in vitro in the mtbc proteome by mawyuenyega et al.,  <dig>  <cit> , that is, is there constitutive expression of the mtbc type iii-a cas <dig> gene?
support for this view comes from findings that cas <dig>  is an essential gene for mtbc growth in vitro .
in contrast, for mtbc h37rv grown in vivo the cas <dig> gene was unessential in an infected-mouse model  <cit>  and the cas <dig> protein was not detected in the proteome in an infected-guinea pig model  <cit> .
perhaps in vivo expression of cas genes is regulated differently than in vitro expression.
in any case, if cas <dig> gene and cas <dig> protein are essential for in vitro growth of mtbc, the deleted-dr strains and beijing family strains  and strains with potentially defective cas <dig> such as t3_eth , must have developed compensatory mechanisms which allowed them to be cultured from clinical samples.
another possibility that has not been examined, is whether cas <dig> expression is somehow connected with possible constitutive expression of crrnas – in p. furiosus the approximately  <dig> crrnas from all  <dig> crispr loci are constitutively expressed , but to our knowledge nothing is known about the expression of mtbc crrnas.
what possible effects might the loss of the cas <dig> gene have on any given mtbc strain?
cas <dig> protein physically and genetically interacts with key components of e. coli dna repair systems, including recb, recc and ruvb, and cas <dig> deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation  <cit> .
is there the possibility of a similar phenotype in the deleted-dr strains of mtbc, or intact-dr strains of mtbc, that are defective in the cas <dig> gene?
there is no data with which to answer this question, but some pertinent observations can be made concerning mtbc.
as already mentioned, the widespread successful mtbc pathogens of the beijing family  <cit>  all lack the cas <dig> gene as part of their defining rd <dig> deletion   <cit> .
evidence presented in this paper showed that all deleted-dr strains  lost at least their cas <dig> and cas <dig> genes , but previously beijing was considered the only natural instance known of crispr-containing bacteria to be without a cas <dig> gene  <cit> .
however this knowledge has been updated by makarova et al.,  <dig>   <cit>  who report that the cas1-cas <dig> adaptation module is dispensable in types iii-a and iii-b crispr-cas systems.
no evidence has been offered for impaired chromosomal segregation in beijing mtbc, but there appears to be an increased sensitivity to dna damage as shown by beijing-specific snps resulting in a potential mutator phenotype   <cit> , and by the fact that in israel  <cit>  and worldwide , mtbc beijing family strains are observed to be robustly transmissible  and to have an increased tendency to be mdr.
thus one could speculate that in beijing, loss of cas <dig> may have contributed to increased adaptability .
in this regard, it is notable that the largest mdr-containing african-origin cluster in israel  <cit>  and some parts of africa  <cit>  is t3_eth, which was shown in this study to have  <dig> potentially disruptive snp missense mutations in its cas <dig> gene .
further studies are necessary to determine whether t3_eth has functional cas <dig>
mtbc strains differ from most other bacteria by lacking a mismatch repair system  <cit> , the lack of which causes instability within genomic regions containing nucleotide repeats  <cit> .
we previously published a possible instance of this instability observed in a large cluster of cas family mtbc in israel  <cit> , and one could speculate that this instability may be one of the drivers of miru-vntr polymorphism in general which is extremely useful in molecular epidemiology of mtbc  <cit> .
however, wanner et al.
<cit>  analyzed the mtbc strain h37rv and other bacteria, and determined that deficiency in dna-repair increases the selection for inherently stable genomes “by using codons to encode proteins in a context-dependent manner that prevents the emergence of nucleotide repeats.” the results reported and summarized in our paper suggest that different families of mtbc have different burdens of dna-repair deficiency , and thus the important observation of stabilization of the h37rv genome may not be generalizable to all mtbc without examining other strains belonging to at least the other major families of mtbc.
the effect of defective dna-repair genes is cumulative: the more defective dna-repair genes, the more mutability  <cit> , which presumably could lead to increased adaptability, with consequent accumulation of mutations in global transcription/stress response genes favoring survivability/persistence, transmissibility, and acquisition of drug resistance.
all mtbc strains, as noted above, lack the mismatch repair system, and it is therefore important to draw attention to the possible cumulative effect in beijing set in motion by the additional loss of cas <dig>  and its putative associated dna-repair network  <cit> , in addition to other known beijing-specific defective dna-repair genes, .
the dna-repair mechanism in mtbc is already known to be one of the slowest in bacteria  <cit> , and cumulative loss of dna-repair functionality could only be expected to make the dna-repair mechanism even slower, exacerbating the acquisition of mutations known to result from defective dna-repair  <cit> .
CONCLUSIONS
deleted-dr strains showed complete loss of clustered regularly interspaced short palindromic repeats  and accompanying spacers, retention of the mid-crispr is <dig> mobile element in clonally specific orientation, and accompanying clonally-specific loss of flanking genes, including adjacent crispr-associated  genes  <cit> .
all deleted-dr strains were accompanied by a loss of at least the cas <dig> and cas <dig> genes.
tentative miru-vntr-based assignments of the deleted-dr isolates to mtbc major spoligotype families a haarlem, b beijing, and c eai were validated by identifying lineage-defining long sequence polymorphisms  known as region of difference  deletions  <cit> , corresponding to major spoligotype families  <cit> .
similarly, the deleted-dr strain srr <dig> whose sequence was assembled from downloaded reads, deleted cas <dig> and cas <dig> and was assigned by rd deletion to the mtbc major spoligotype family cas.
since cas <dig> deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation  <cit> , we discussed the possibility of a similar phenotype in the deleted-dr strains of mtbc and in the intact-dr mtbc beijing that are defective in the cas <dig> gene , and in the intact-dr mtbc t3_eth that are potentially defective in the cas <dig> gene .
additional files
additional file 1: table s <dig>  miru-vntr loci and octal spoligotypes.
samples: a-b-c&t3_eth.
controls: 0486– <dig> beijing clinical isolate, ecdcpt 21& <dig> eai, reference h37rv, pasteur bcgp <dig>
additional file 2:
list s <dig>  sources for drug sensitivity data presented in table  <dig>
additional file 3:
sequences s1–s <dig>
additional file 4: table s <dig>  location of crispr-flanking genes and mid-crispr is <dig> mobile element used for primer choices and alignments.
additional file 5: table s <dig>  primers employed for pcr verification of deletions of crispr/dr and flanking regions.
additional file 6: figure s <dig>  visualization of mid is <dig> orientation: blue for wild type  or for unknown orientation; red for inverse orientation .
dr plus flanking sequences were aligned to dr and flanking regions of reference template h37rv.
sequences were from mycobacterium tuberculosis ngs read sets generated in this study , on-line resources of read sets, mapped on h37rv nc_ <dig> , and on-line resources of complete chromosome sequences.
additional file 7: figures s2–s <dig>  mutations of cas <dig> shared by mtbc t3_eth strains and by proto-beijing strains.
abbreviations
cascrispr-associated
cascentral asian
cascrispr-associated gene
crisprclustered regularly interspaced short palindromic repeats
drdirect repeat region
drplussequence of dr plus flanking regions – about  <dig> kbp
lsplong sequence polymorphism
miru-vntrmycobacterial interspersed repetitive units – variable number tandem repeats
mtbc
mycobacterium
tuberculosis complex
ngsnext generation sequencing
rdregion of difference
spoligotypingspacer oligonucleotide typing
we thank the staff of the national mycobacterium reference center for routine excellent performance of mycobacterium tuberculosis growth, identification and drug sensitivity tests, and maintenance of the mycobacterium tuberculosis strain repository.
funding
this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
availability of data and materials
ngs sequence reads for the  <dig> deleted-dr mtbc isolates from patients a haarlem, b beijing, and c eai and for  <dig> intact-dr mtbc t3_eth, have been deposited in the international dna database genbank under bioproject prjna <dig>  accession no.
srp <dig>  all other datasets supporting the results of this article are available in the main paper and additional files, or by downloading from international databases such as genbank.
authors’ contributions
pjf, in, dg, and er conceived and designed the study.
pjf, in, al, srh, and ls conducted the molecular work and data analysis.
ls constructed a method to obtain spoligotypes from ngs reads.
pjf constructed an application employing bionumerics  and excel  to obtain spoligotypes from ngs reads and complete whole genome sequences.
pjf, in, dg, and er drafted the manuscript.
pjf, dg, and er coordinated the study.
er, hk, zd and dc contributed to the development of the project.
all authors have read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval and consent to participate
no ethics approval was required for the public sequence data used in this study.
no ethics approval was required for the anonymous use of mycobacterium tuberculosis strains stored in the mycobacterium tuberculosis repository of the national mycobacterium reference center.
agreement to be a co-author
all authors have agreed to be co-authors of this article: “structure and variation of crispr and crispr-flanking regions in deleted-direct repeat region mycobacterium tuberculosis complex strains”.

 the national public health laboratory tel aviv, national mycobacterium reference center routinely genotypes all new culture-positive tuberculosis cases in israel with spacers reverse line blot spacer oligonucleotide typing, and loci mycobacterial interspersed repeat units – variable number tandem repeats typing using multiplex pcr and capillary electrophoresis. the results are stored in excel, bionumerics, and whonet databases. from 2008– over strains were genotyped and tested for drug sensitivity to first line, and when necessary, second line drugs. six drug-sensitive isolates from three different patients a, b, and c yielded negative spoligotyping results and three different loci miru-vntr profiles respectively. variation in the structure of crispr and crispr-flanking regions of these negative spoligotype mtbc isolates was observed when ngs reads were mapped on reference whole genome sequence h37rv nc_. this yielded visualization of coverage. after substitution of manual-pcr-derived dr-spanning sequences for the corresponding map-derived regions, further details of structural variation were revealed by alignments of annotated subsequences containing crispr plus crispr-flanking regions to the annotated reference h37rv sequence.fig location and coverage of h37rv-mapped ngs-reads of negative-spoligotype strains a, b, c and intact-dr t3_eth fig structure and variation of crispr and crispr-flanking regions in deleted-dr and intact-dr mtbc in addition to the drug-sensitive deleted-dr isolates, two drug sensitive and multiple-drug-resistant intact-dr strains from t3_eth, our largest african-origin mdr-containing cluster, were analyzed by ngs. these provided positive controls for the sensitivity of our methods to correctly characterize from ngs data, the crispr-cas regions of intact-dr mtbc, especially with respect to the presence or absence of crispr spacers.table characteristics of ngs sequenced deleted-dr sample strains a, b, c and intact-dr t3_eth, and whole genome sequences obtained on-line or assembled from on-line read sets# mutt4 t2 ogt #id, sample, read set or ncbi accession number; octal, from spoligosome sit, spoligotype international type and associated family; ns, no sit; drug res, resistant to drug, i-isoniazide, r-rifampicin, e- ethanbutol, s-streptomycin, p-pyrazinamide, ds: none, dr: at least one but not i&r together: mdr: at least i&r, xdr: at least mdr plus defined 2nd line drugs consisting of at least quinolone and at least one of the injectable drugs kanamycin, capreomycin or amikacin, tdr: resistant to all 1st and 2nd line drugs. a-g: additional file 2: list s – sources of drug sensitivity data; wt, identical to reference genome h37rv nc_; crispr is orientation of the is mobile element found in the direct repeat region, wt: same orientation as in h37rv, inv: inverted, unk: unknown; mutation in mutt, codon 48: arg cgg ggg gly; mutation in mutt, codon 58: gly gga cga arg; mutation in ogt, silent codon 12: gly ccc tcc gly, missense codon 15: thr ggt gct ser; regions of difference rd, 105: proto and all modern beijing, 207: lsp in crispr region defining modern beijing – includes cas gene deletions, 181-150-142: polymorphic lsps for beijing, 182: marker for haarlem, 239: marker for eai, 750: marker for cas complete loss of all direct repeat region repeats and spacers, negative spoligotyping results, and loss of genes in flanking regions has been reported previously for mtbc with negative spoligotypes identical to those currently assigned to sit and for other bacteria, however, the molecular structures of the crispr regions and flanking crispr-associated regions were not reported, except for a comparison to the homologous genomic region in non-tuberculous mycobacteria. the evidence reported herein clearly showed that the deleted-dr and accompanying deleted crispr-flanking genes of the mtbc described in this study were not an instance of the orfs absent in ntm species. also, this study is the first known to us to employ identification of region of difference deletions in order to assign negative spoligotype mtbc to their corresponding major spoligotype families. the mtbc rd deletions are genomic long sequence polymorphisms which were detected in this study by aligning mycobacterium tuberculosis complete genome sequences with the mtbc reference strain h37rv complete genome sequence. the mtbc rd deletions, and also an extensive set of single nucleotide polymorphisms, have been shown by coll et al. in an on-line available application for analysis of ngs reads, to have phylogenetic behavior that corresponds well with the phylogenetic distribution obtained by analysis of 43-spacer spoligotypes. 43-spacer spoligotypes have been assigned type numbers and family names. the 43-spacer spoligotypes are more informative than the rd types and snp types, thus any given specific 43-spacer spoligotype cannot be inferred from an rd type or snp type, but the spoligotype family can be inferred from rd or snp information, and in addition some sub-lineage information can be obtained from the snp type. therefore, when one encounters a negative spoligotype by conventional screening for the crispr spacers, if ngs sequencing can be performed for the deleted-dr strain, two convenient ways exist to determine the spoligotype family: 1) map the ngs reads to reference h37rv, align the constructed mtbc sequence with the h37rv sequence, and search for the relevant rd deletions, and 2) submit the ngs reads to the coll et al. site for analysis of rd deletions and snps. the rd data shown in table  suggested that a third way could be developed which would not require sequencing for ngs reads: a possible multiplex pcr probe for relevant rd deletions, which could be analyzed by fragment analysis as in miru-vntr typing, or by real time pcr. various methods have been proposed for spoligotyping from ngs reads, but all involve detection of crispr spacers and thus would only yield negative results with our deleted-dr negative spoligotype strains. a combination of the pcr-based methods spacer spoligotyping and loci miru-vntr typing has replaced rflp-typing as the mtbc genotyping screen of choice, due to the speed and convenience of the pcr-based methods, genotype discrimination as good as or better than that for rflp-typing, and the ability to store the pcr-based results in easily manipulated digital format. on a population level, screening for spoligotype family provides insight into the bio-geographic burden of tb in the world and for any particular country. thus it is useful to be able to determine the spoligotype family for a strain that has a negative spoligotype by conventional screening. while in general the more informative loci miru-vntr type will still phylogenetic-wise segregate according to its complementary spoligotype, for certain miru-vntr profiles the miru-spoligo correlation may be difficult to establish. a published example of this showed that the miru-vntr profiles in spoligotype families cas and beijing can sometimes be difficult to distinguish. in another case, very similar miru-vntr profiles were found in both the t and lam spoligotype families. in these cases it was not only useful, but indeed necessary, to determine at least the spoligotype family. on a clinical level, the combination of spoligotype and miru-vntr type complements and guides classical epidemiological investigation to clarify questions of transmission between individuals, transmission chains among larger groups of individuals, questions about whether a recurrence of tb is due to reactivation or reinfection, and resolution of questions arising when a lab provides a positive tb report that is inconsistent with the physician's observation – that is- the possibility of a false positive due to lab contamination. often loci miru-vntr alone supplementing classical epidemiology is enough to resolve these issues. nevertheless, if the strains involved have negative spoligotypes on conventional screening, knowledge of at least the spoligotype family can help validate the results concluded from classical epidemiology supplemented with miru-vntr typing. finally, there are often gaps in epidemiological knowledge when trying to establish transmission chains among larger groups of individuals. these knowledge gaps can be exacerbated by subtle polymorphisms in miru-vntr profile, where knowing only the spoligotype family will not be enough to firmly establish the transmission chain. therefore, when confronted with negative spoligotypes in large putative transmission chains, it seems reasonable to recommend that all the strains should be processed for ngs reads. then the ngs reads for the negative spoligotype strains should be used for spoligotype family determination, and subsequently for snp clonality-check comparisons among the strains to delineate the outbreak. mycobacterium tuberculosis complex bacteria possess a type iii-a crispr-cas system structurally homologous to type iii-a of other genera. active type iii-a systems provide adaptive immunity to bacteriophage and plasmids, and perform endogenous roles, for example, endogenous dna repair via cas endonuclease. in mtbc the crispr region is also known as the direct repeat region. polymorphism in the number and type of spacers in the dr/crispr region of any given mtbc strain, has been and continues to be extensively used for molecular epidemiology of mtbc, especially for overviews of phylogenetic and bio-geographical distributions of clades. fourty three-spacer-spo-plus-24-loci-miru genotyping provides molecular epidemiology at the resolution of clonal transmission, with the exception of certain instances, for example, of convergent evolution. the functional activity of the mtbc crispr-cas system remains to be investigated, but the highly conserved sequence and order of the crispr/dr region spacers which is the basis for the epidemiological and phylogenetic usefulness of polymorphism on spacer oligonucleotide typing, has been interpreted to imply at least partial inactivity of the mtbc crispr-cas system. this interpretation presumes that the insertion of new spacers in the crispr/dr region is one of the fundamentals of, and evidence for, adaptive immunity, and thus lack of evidence for insertion of new spacers in mtbc implies at least partial inactivation of the crispr-cas adaptive immune system in mtbc. the mtbc type iii-a crispr-cas system is defined by the presence of cas10/csm and csm accessory genes, and is predicted to be in some manner active on the basis of the presence of intact cas and cas genes. in addition, some sort of activity even if only in endogenous roles, can be predicted from genbank annotation that informs of mass spectroscopy support for the existence of most of the cas gene products, including cas1in the reference mtbc strain h37rv. the lineage-defining rd deletion of the super-spreader mdr-containing beijing family includes complete deletion of cas and cas genes, so possession of a fully active type iii-a crispr-cas system is not a prerequisite for a successful mtbc lineage. interestingly, the complete loss of crispr spacers in e. coli gave the same dna-repair deficient phenotype as deletion of the e. coli cas gene. using m. smegmatis as a model for mtbc, cas by itself was shown to be deleterious to mycobacterium survival, thus it may be significant that deletion of cas was always accompanied by deletion of at least cas in summary, the results showed that complete deletion of dr region repeats and spacers in negative-spoligotype mtbc was accompanied by clonal-specific loss of flanking genes, always accompanied by complete deletion of cas complete deletion of putative dna-repair gene cas and retention of the middle is mobile element. for the first time it was shown that negative spoligotype mtbc nevertheless could be assigned to major spoligotype families by identification of ngs-revealed corresponding lineage-defining rd deletions. since there was only deleted-dr strain-type per rd-defined family, there was not enough information to decide if the loss of cas genes was solely clonally specific, or perhaps had lineage-defining features. methods growth of mycobacterium tuberculosis strains, and drug sensitivity tests all approximately new culture-positive mtbc strains collected during 2008– were analyzed. cultures were grown and identified as mtbc as previously described. drug sensitivity tests were performed as previously described. extraction of mtbc dna preparations of rflp-grade mtbc dna for ngs sequencing, and of “crude” sonicated, heated extract for genotyping, were done as previously described. genotyping of mtbc dna routine genotyping of mtbc dna by spacer spoligotyping and loci miru-vntr typing were done as previously described. bcgp was the generous gift of professor hillel bercovier, hebrew university of jerusalem. european communicable disease center proficiency test samples and and ecdcpt h37rv control dna were obtained originally as proficiency test materials. genotyping results were stored in an excel database, a bionumerics ver database, and a whonet database. ngs assay and bioinformatics analyses at the scripps research institute, including 1) coverage-curves illustrating deletion of crispr/dr-cas regions present in the template reference h37rv chromosome sequence, and 2) determination of spoligotypes from ngs reads rflp-grade dna samples were sent to mgr. steven robert head and mgr. lana schaffer of the scripps research institute in san diego, california usa for ngs and draft bioinformatics analysis. one microgram of each sample was sheared on the covaris s each sample was then end repaired, a-tailed and adapter ligated. the samples were cleaned with agencourt beads and treated with 6 cycles of pcr. after pcr the samples were cleaned using zymo columns and loaded on a 2% agarose gel. the gel was cut from 400–700 bp and cleaned again with the zymo column. the samples were sequenced on an illumina miseq system as paired-end reads each with length 300 bp. the alignment was performed using novoalign v. without soft clipping to allow reads which cross the ends of the reference sequence as would be the case of a circular genome. the mycobacterium tuberculosis reference sequence used for the alignment was mtb-h37rv-asm19595v and for repeat sequences not in the mapped-to-h37rv mtbc sequence was mycobacterium bovis af2122/ the reads were preprocessed by trimming adaptor sequences, trimming by base call quality scores lower than removing unmapped reads and duplicate reads, and removal of reads with mapping quality score less than one. repeat sequence coordinates and sequences were taken from the jansen paper. for coverage curves illustrating deletion of crispr-dr-cas regions that were present in the template h37rv chromosome sequence, the aligned bam files were converted to wig files using bamtools, bedtools, and ucsc-tools. the wig files were imported into ucsc microbial genome viewer. recovery of spoligotypes from ngs data was accomplished as follows. a bed file was created using the coordinates of the dr repeat units. this bedfile and the aligned bam files were used with “samtools bedcov” tool to output number of counts for each repeat unit. in order to produce a binary table matching the spoligotyping regions with less than counts were considered absent. bioinformatics at the national public health laboratory tel aviv analyses of our ngs raw data were accomplished by use of bionumerics version and import and processing of 1) cleaned paired-end and single-end fastq reads received from scripps, 2) reads formatted as sra and fastq files downloaded from ncbi or other sources, and 3) fastq or embo/gb format reference whole chromosome sequences or contributed chromosome sequences from ncbi or other sources. for visualization of the molecular details of the crispr-dr and flanking regions, reads were resequenced/mapped onto reference h37rv nc_ using default parameters with the following changes: accept paired-end reads, minimum coverage minimum sequence length before considered for contig 500 nt. the “coverage” was examined as percentage of the h37rv genome matched, and as the number of reads contributing to each base pair assignment. the mapped chromosomes were annotated with h37rv genbank and bionumerics-predicted features and matrix compared and aligned in chromosome comparison mode using default parameters. for decisions about reading frames and codon assignments, reference h37rv nc_ annotated only with genbank annotation was included in comparisons. default parameters were: i. comparison project type: full sequence based; matrix: matrix; self-comparison of sequences; direct sequence orientation; inverted sequence orientation; seed extension dna seed: minimum matches in window size of bases; amino acid seed: minimum matches in window size of amino acids; stretch extension minimum matches in window size of bases; minimal stretch length: 50; ii. seeds seed database: seed type: dna, length:5; seeds used in project: seed type: transl, length: and iii. alignment settings template entry: usually the gb plus bn annotated reference h37rv; stretch import minimum length: 0; minimum identity: 0; stretch mapping open gap penalty: 10; extend gap penalty 10; do not allow repetition of mapped stretches; full alignment of stretch ends: checked; full alignment settings: open gap penalty: 100; extend gap penalty: 0; use fast algoritm: checked; k-tuple size:2; no. of diagonals: this generated a schematic suitable for visualization of deletions, while choosing “show text view” while in alignment sub-mode in the comparison mode allowed visualization of single nucleotide polymorphisms. an important point is that any given set of parameters has its own strengths and limitations. the default parameters of bionumerics and chromosome comparison did not detect inverse is in the crispr-cas region, nor were they suitable for comparison of shorter sequences. the region containing the crispr-cas sequence plus flanking regions was cut out, annotated and used for further comparisons in the alignment window when high similarity was expected, or in the chromosome comparison mode and alignment sub-mode with appropriate parameters when some dissimilarity was expected. the appropriate parameters for comparison of shorter sequences in the chromosome comparison mode were: i. comparison seed extension: dna seed: minimum matches in window size of bases; amino acid seed: minimum two matches in window size of amino acids; stretch extension: minimum matches in window size of bases; minimal stretch length: 12; ii. seeds seed database: seed: 111; type: dna; length: 3; seeds used in project: seed: 111; type: dna; length: 3; iii. alignment settings: full alignment settings: open gap penalty: 0; no. of diagonals: other parameter sets can be better for other uses, and must be determined empirically. in order to obtain contig-corrected sequences for the six approximately kbp crispr plus crispr-flanking subsequences of the deleted-crispr strains from patients a, b, and c, the ngs-obtained h37rv-mapped subsequences containing the crispr-spacers regions were excised from the assembled mapped whole genome sequence at predetermined flanking genes on each side, and replaced with sequences of pcr validated contigs spanning the crispr-spacers-dr region, that is, with 5’ and 3’ ends of the contigs overlapping the conserved crispr-flanking genes characteristic for each deleted-dr strain. these “drplus” sequences corrected for the appropriate contigs are found in additional file 3: sequences s1–s the crispr-flanking genes and their locations are listed in additional file 4: table s the pcr validated contigs were constructed using primers inferred from the ngs data from mapped to h37rv, and de novo, sequences according to which regions could be expected to be “conserved” in the crispr-spacers region itself, and in the immediately adjacent crispr-flanking regions characteristic for each deleted-dr strain. pcr and agarose gel electrophoresis were performed as previously described and the pcr product purified on qiaquick columns. the purified pcr product was sent for forward and reverse sequencing to a commercial sequencing facility, and the sequences from the ab sequencing files were trimmed and used to construct contigs spanning the crispr-spacers region. validation of sequence trimming and alignment for contig construction was done in parallel using a different bioinformatics tool. the h37rv nc_ whole genome sequence locations, corresponding to the dr plus flanking regions of the mapped deleted-dr strains a, b, and c, were used for alignments for primer construction. the major primers used for manual pcr were as shown in additional file 5: table s recovery of spoligotypes from ngs reads, or from downloaded reference or contributed chromosomes, was accomplished by constructing a new bionumerics version application, manuscript in preparation. in addition, the sequences for the dr region-spanning contigs for deleted-dr strains a, b, and c were examined by ncbi blast for sequences corresponding to spacer sequences. drug sensitivity and lineage information for downloaded reads was obtained either from the literature or by uploading the reads to tb profiler which reported back the drug sensitivities for first and second line drugs, and projected lineages. rd deletion confirmation of lineages was obtained from bionumerics chromosome comparison alignments of complete genome sequences to the reference h37rv sequence.  variation in the crispr and crispr-flanking regions: clonal specific deletion of crispr-flanking regions in deleted-dr strains – including deletion of at least cas and cas and retention of the is mobile element in deleted-dr strains in fig.  coverage of the homologous reference h37rv sequences was approximately reads for each isolate, with the negative spoligotype isolates a, b, and c showing wide gaps to the left and right of the middle sequence. the middle read-coverage length was suggestive of that of an is mobile element. the coverage of the template h37rv genome ranged from to % for the deleted-crispr-spacers-cas strains a, b, and c, and to % for the t3_eth strains. variations in the crispr and crispr-flanking regions of negative spoligotype mtbc isolates from patients a, b, and c were observed in the visualization of ngs reads mapped on reference h37rv nc_ and annotated alignments of drplus subsequences to the drplus reference h37rv subsequence. for comparison, intact-dr results from three ngs-sequenced t3_eth were shown. drplus subsequences from isolates a, b, c, and isolate srr which was assembled from downloaded reads, were aligned to reference template h37rv subsequence. a, b, and c – specific deletions of crispr-flanking genes were observed, including deletions of cas genes to the right of the retained mid is all deleted-dr strains, retained their mid is mobile element, and deleted at least cas and cas. the deleted-dr b isolates showed a right-side deletion of the same size and location as that shown in beijing-family strains. subsequent rd deletion identification of deleted-dr b isolates as beijing, suggested that the b deletion may be an extension of the beijing lineage-defining rd deletion. this was further suggested by the fact that the deleted-dr b isolates shared the beijing-specific mutator phenotype snps mutt4|t2|ogt. thus we propose to name the deletion seen in the deleted-dr b isolates as “extended rd207” in accordance with the terminology adopted by luo et al. for the rd deletion in proto-beijing: extended rd. it was interesting, but of as yet unproven important significance as discussed below, that in contrast to the inverse orientation of the is mobile element in the dr region of many if not all validated beijing-family sequences the orientation of is in the beijing deleted-dr b isolates was wild type, that is, the same as for reference template h37rv. validation of retention of mid-is mobile element, and of loss of varying amounts of genomic regions flanking the deleted crispr/dr regions of isolates a, b, and c in order to validate retention of is and clonally specific loss of flanking genomic sequences in deleted-dr isolates a, b, and c, primers were chosen from conserved flanking genes and the mid is and used to generate amplicons by manual pcr which were assembled into contigs for a dr-spanning sequence for each isolate a, b, and c. these validated dr-spanning sequences were substituted into the drplus sequences of h37rv-mapped isolates a, b, and c, resulting in a “corrected’ drplus sequence for each isolate a, b, and c. alignment of the corrected drplus sequences to template h37rv confirmed that mid-is was retained in each deleted-dr isolate a, b, and c. it was interesting that the orientation of this is of each beijing deleted-dr isolate b was wild type with respect to h37rv, and inverse with respect to the orientation of is observed in reference whole genome beijing sequences. alignment of the manual pcr corrected dr plus flanking sequences to template h37rv also confirmed the clonally specific loss of crispr-flanking regions, as shown in fig.  and detailed in additional file 4: table s each deleted-dr isolate a, b, and c showed deletion of at least cas and cas from among the cas genes.table the presence or absence of mutations in crispr-associated genes in mycobacterium tuberculosis complex strains compared to reference template h37rv nc_ 4411532 bp. the mutations are listed in ascending order of their location relative to the reference h37rv chromosome cas2 cas1 cas1 cas1 cas1 csm6 csm6 csm6 csm6 csm6 csm6 csm5 csm5 csm5 csm4 csm4 csm4 csm4 nonea csm3 nonea csm2 cas10/csm1 cas10/csm1 cas10/csm1 cas10/csm1 cas10/csm1 nonea cas6 anone: no mutations listed for the strains examined; location: base pair of h37rv nc_ where snp can be found, or base pair approximately in the center of a given deletion or insertion; ssp short sequence polymorphism, snp single nucleotide polymorphism the absence of mutations in csm csm and cas could mean that those genes cannot tolerate mutations due to the importance of their sequence, or transcription or translation products. however, mutations could appear if a larger set of strains were examined, a possibility suggested by the recent report that the cas gene is dispensable in type iii crispr-cas systems. did the retained is mobile element have a role in generating the crispr and flanking-sequences deletions observed in the deleted-dr strains? the mobile element is inserts into the mycobacterium tuberculosis genome in a variety of ways with a variety of potential effects, some of which we have discussed previously. the dr/crispr region is a hot spot for is insertions, where there can be or more is elements inserted in the dr region. in this study all the mtbc strains examined had mid dr is mobile element. after crispr deletion, along with deletion of varying amounts of dr-flanking sequences, the dr region was left with a residual is mobile element attached to what remained of the flanking sequences. the is mobile element has the ability to delete sequences on either side of it. in the mtbc beijing family, the mid dr is mobile element is found in reverse orientation to the h37rv mid dr is sequence. the results shown in this paper were consistent with these previous findings, as except for the deleted-dr beijing strains from patient b, all beijing mid dr is elements from validated complete chromosome sequences, had orientations inverse to the mid dr h37rv is orientation. in the deleted-dr beijing strains from patient b, the residual mid dr is was oriented in the same direction as the reference h37rv mid dr is mobile element. we speculate that generation of the deleted-dr extended rd deletion involved this is mobile element, which in the process was “flipped” from its previous characteristic beijing inverse orientation back to a wild type h37rv-like orientation. the mechanism behind the is ability to generate deletions has been investigated, but to the best of our knowledge this issue has not been resolved. in summary, it is probable that the residual is mobile element found attached to the presumed dr flanking sequences of the deleted-dr strains, played an as yet uncharacterized role in the deletion of the crispr sequences and flanking genes observed to be deleted in the deleted-dr strains. validation of total loss of crispr spacers in deleted-dr isolates a, b, and c, and in downloaded read-assembled deleted-dr strain srr1188186 approach 1 recovery of spoligotypes from ngs data for isolates a, b, and c, and t3_eth, was accomplished as follows. a bed file was created using the coordinates of the dr repeat units. this bedfile and the aligned bam files were used with “samtools bedcov” tool to output number of counts for each repeat unit. in order to produce a binary table matching the spoligotyping, regions with less than counts were considered absent. by this method, the correct spoligotypes could be obtained for the intact-dr t3_eth. approach 2 in order to validate the total loss of spacers and repeats in the deleted-dr isolates, for each isolate a, b and c corrected drplus sequences were examined for spacer and repeat sequences, which were found to be totally absent from these corrected drplus sequences. approach 3 in order to further confirm the total loss of crispr spacers in deleted-dr isolates a, b, and c, and in downloaded read-assembled deleted-dr strain srr a new application was developed for bionumerics and and excel. the application yielded results consistent with other approaches in that no spacers were detected in deleted-dr strains, and correct spoligotypes were assigned to reference h37rv, reference bcg, and the sequenced t3_eth. crispr-cas defects: comparison of mtbc strains for insertions/deletions and snps in cas genes since isolates from patient b were beijing, it was of interest that the deleted-dr deletion consisted of a left-side-of-is extension of the beijing-defining rd deletion. the rd deletion is found in all modern beijing, and removes most crispr-spacers and cas genes through rv. table  lists the cas gene mutations in order of ascending location on the h37rv chromosome, and the corresponding mutated genes for the strains examined in this study. notable mutations were the extensive deletions of cas genes in beijing family members including deletion of cas; the deletion of cas and cas by all deleted-dr strains; t3_eth specific missense snps in cas1; and a proto-beijing specific missense snp in cas. t3_eth is our largest mdr-containing african-origin cluster, and has been reported by others to have proclivity for mdr. how likely is it that the cas proteins from type iii-a systems in mtbc also have a role in dna repair in a manner similar to that of cas proteins from type i-e in e. coli? babu et al. induced dna damage in e. coli by exposure to mitomycin c, uv light or cisplatin. they noted, and gave examples, that cas proteins from different genera of bacteria can display related but different biochemical properties. thus any given cas protein must be characterized for its biochemical and functional roles, which they did for the e. coli type i-e cas but which has not yet been done for the mtbc type iii-a cas moreover, we and others were not able to find any studies done in any genus to determine the biochemical and functional roles of type iii systems. nevertheless, makarova et al. noted that the cas family is the most conserved cas protein family and showed that the cas phylogenetic tree corresponds nicely with the phylogeny of the bacterial genera. by use of the ncbi tool cdart, we observed that the e. coli type i-e cas had homologous domain architecture with the m. tuberculosis type iii-a cas the cas proteins from both were members of the cas endonuclease super-family cas1_i-ii-iii. only the e. coli cas was labeled “multifunctional” because there is no relevant data yet for m. tuberculosis cas however, we consider it likely that the mtbc cas will eventually be shown to have a role in endogenous dna repair. it was noted by makarova et al., that the adaptation module is dispensable in subtypes iii-a and iii-b. this would tend to support the idea that cas in mtbc is nonessential, even if it does have an endogenous dna-repair activity role. consistent with this, the beijing family of mtbc has deleted cas and cas, yet still is a very successful pathogen. however, nonessential does not necessarily mean non-important. cas is produced in mtbc, except for the possible exceptions noted in our paper, and its absence or mutation-induced defectiveness could in theory result in impaired dna repair and consequent increased mutability. has induction of the cas gene been observed in response to dna-damage in genera other than that of e. coli which shows type i-e cas induction? pyrococcus furiosus has different crispr loci and different crispr-cas systems, type iii-b, type i-g, and type i-a. williams et al., analyzed the transcriptome of p. furiosus after induction of dna damage by gamma irradiation. they observed genes that had up to a 10-fold increase in mrna levels following gamma irradiation. of the genes were crispr-associated, but none of them was cas although was predicted to be involved in dna repair and recombination. interestingly, all up-regulated crispr-associated genes belonged to the type i-a system, and none of the genes in the other systems, type iii-b and type i-g, were similarly up-regulated. williams et al., note that some efficient dna repair pathways appear to be constitutively expressed in the hyperthermophilic archaeon p. furiosus, which they attribute to the result of life at high temperature. to our knowledge there are no studies of induction of cas in response to dna-damage, other than the report of induction of cas in e. coli. life in a macrophage, and frequent exposure to hypoxic conditions, also lead to a stressful environment for m. tuberculosis, and we ask if this is the reason that cas was found in vitro in the mtbc proteome by mawyuenyega et al.,, that is, is there constitutive expression of the mtbc type iii-a cas gene? support for this view comes from findings that cas is an essential gene for mtbc growth in vitro. in contrast, for mtbc h37rv grown in vivo the cas gene was unessential in an infected-mouse model and the cas protein was not detected in the proteome in an infected-guinea pig model. perhaps in vivo expression of cas genes is regulated differently than in vitro expression. in any case, if cas gene and cas protein are essential for in vitro growth of mtbc, the deleted-dr strains and beijing family strains and strains with potentially defective cas such as t3_eth, must have developed compensatory mechanisms which allowed them to be cultured from clinical samples. another possibility that has not been examined, is whether cas expression is somehow connected with possible constitutive expression of crrnas – in p. furiosus the approximately crrnas from all crispr loci are constitutively expressed, but to our knowledge nothing is known about the expression of mtbc crrnas. what possible effects might the loss of the cas gene have on any given mtbc strain? cas protein physically and genetically interacts with key components of e. coli dna repair systems, including recb, recc and ruvb, and cas deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation. is there the possibility of a similar phenotype in the deleted-dr strains of mtbc, or intact-dr strains of mtbc, that are defective in the cas gene? there is no data with which to answer this question, but some pertinent observations can be made concerning mtbc. as already mentioned, the widespread successful mtbc pathogens of the beijing family all lack the cas gene as part of their defining rd deletion. evidence presented in this paper showed that all deleted-dr strains lost at least their cas and cas genes, but previously beijing was considered the only natural instance known of crispr-containing bacteria to be without a cas gene. however this knowledge has been updated by makarova et al., who report that the cas1-cas adaptation module is dispensable in types iii-a and iii-b crispr-cas systems. no evidence has been offered for impaired chromosomal segregation in beijing mtbc, but there appears to be an increased sensitivity to dna damage as shown by beijing-specific snps resulting in a potential mutator phenotype, and by the fact that in israel and worldwide, mtbc beijing family strains are observed to be robustly transmissible and to have an increased tendency to be mdr. thus one could speculate that in beijing, loss of cas may have contributed to increased adaptability. in this regard, it is notable that the largest mdr-containing african-origin cluster in israel and some parts of africa is t3_eth, which was shown in this study to have potentially disruptive snp missense mutations in its cas gene. further studies are necessary to determine whether t3_eth has functional cas mtbc strains differ from most other bacteria by lacking a mismatch repair system, the lack of which causes instability within genomic regions containing nucleotide repeats. we previously published a possible instance of this instability observed in a large cluster of cas family mtbc in israel, and one could speculate that this instability may be one of the drivers of miru-vntr polymorphism in general which is extremely useful in molecular epidemiology of mtbc. however, wanner et al. analyzed the mtbc strain h37rv and other bacteria, and determined that deficiency in dna-repair increases the selection for inherently stable genomes “by using codons to encode proteins in a context-dependent manner that prevents the emergence of nucleotide repeats.” the results reported and summarized in our paper suggest that different families of mtbc have different burdens of dna-repair deficiency, and thus the important observation of stabilization of the h37rv genome may not be generalizable to all mtbc without examining other strains belonging to at least the other major families of mtbc. the effect of defective dna-repair genes is cumulative: the more defective dna-repair genes, the more mutability, which presumably could lead to increased adaptability, with consequent accumulation of mutations in global transcription/stress response genes favoring survivability/persistence, transmissibility, and acquisition of drug resistance. all mtbc strains, as noted above, lack the mismatch repair system, and it is therefore important to draw attention to the possible cumulative effect in beijing set in motion by the additional loss of cas and its putative associated dna-repair network, in addition to other known beijing-specific defective dna-repair genes,. the dna-repair mechanism in mtbc is already known to be one of the slowest in bacteria, and cumulative loss of dna-repair functionality could only be expected to make the dna-repair mechanism even slower, exacerbating the acquisition of mutations known to result from defective dna-repair.  deleted-dr strains showed complete loss of clustered regularly interspaced short palindromic repeats and accompanying spacers, retention of the mid-crispr is mobile element in clonally specific orientation, and accompanying clonally-specific loss of flanking genes, including adjacent crispr-associated genes. all deleted-dr strains were accompanied by a loss of at least the cas and cas genes. tentative miru-vntr-based assignments of the deleted-dr isolates to mtbc major spoligotype families a haarlem, b beijing, and c eai were validated by identifying lineage-defining long sequence polymorphisms known as region of difference deletions, corresponding to major spoligotype families. similarly, the deleted-dr strain srr whose sequence was assembled from downloaded reads, deleted cas and cas and was assigned by rd deletion to the mtbc major spoligotype family cas. since cas deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-dr strains of mtbc and in the intact-dr mtbc beijing that are defective in the cas gene, and in the intact-dr mtbc t3_eth that are potentially defective in the cas gene. additional files additional file 1: table s miru-vntr loci and octal spoligotypes. samples: a-b-c&t3_eth. controls: 0486– beijing clinical isolate, ecdcpt 21& eai, reference h37rv, pasteur bcgp additional file 2: list s sources for drug sensitivity data presented in table  additional file 3: sequences s1–s additional file 4: table s location of crispr-flanking genes and mid-crispr is mobile element used for primer choices and alignments. additional file 5: table s primers employed for pcr verification of deletions of crispr/dr and flanking regions. additional file 6: figure s visualization of mid is orientation: blue for wild type or for unknown orientation; red for inverse orientation. dr plus flanking sequences were aligned to dr and flanking regions of reference template h37rv. sequences were from mycobacterium tuberculosis ngs read sets generated in this study, on-line resources of read sets, mapped on h37rv nc_, and on-line resources of complete chromosome sequences. additional file 7: figures s2–s mutations of cas shared by mtbc t3_eth strains and by proto-beijing strains. abbreviations cascrispr-associated cascentral asian cascrispr-associated gene crisprclustered regularly interspaced short palindromic repeats drdirect repeat region drplussequence of dr plus flanking regions – about kbp lsplong sequence polymorphism miru-vntrmycobacterial interspersed repetitive units – variable number tandem repeats mtbc mycobacterium tuberculosis complex ngsnext generation sequencing rdregion of difference spoligotypingspacer oligonucleotide typing we thank the staff of the national mycobacterium reference center for routine excellent performance of mycobacterium tuberculosis growth, identification and drug sensitivity tests, and maintenance of the mycobacterium tuberculosis strain repository. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. availability of data and materials ngs sequence reads for the deleted-dr mtbc isolates from patients a haarlem, b beijing, and c eai and for intact-dr mtbc t3_eth, have been deposited in the international dna database genbank under bioproject prjna accession no. srp all other datasets supporting the results of this article are available in the main paper and additional files, or by downloading from international databases such as genbank. authors’ contributions pjf, in, dg, and er conceived and designed the study. pjf, in, al, srh, and ls conducted the molecular work and data analysis. ls constructed a method to obtain spoligotypes from ngs reads. pjf constructed an application employing bionumerics and excel to obtain spoligotypes from ngs reads and complete whole genome sequences. pjf, in, dg, and er drafted the manuscript. pjf, dg, and er coordinated the study. er, hk, zd and dc contributed to the development of the project. all authors have read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate no ethics approval was required for the public sequence data used in this study. no ethics approval was required for the anonymous use of mycobacterium tuberculosis strains stored in the mycobacterium tuberculosis repository of the national mycobacterium reference center. agreement to be a co-author all authors have agreed to be co-authors of this article: “structure and variation of crispr and crispr-flanking regions in deleted-direct repeat region mycobacterium tuberculosis complex strains”.
we report for the first time negative spoligotype-associated total loss of crispr region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least crispr-associated genes cas and cas since cas deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-dr strains and beijing family strains as both lack the cas gene. two isolates of each of three negative spoligotype mtbc were subjected to next generation sequencing. ngs reads from negative spoligotype mtbc mapped to reference h37rv nc_ suggested that the dr/crispr regions were completely deleted except for retention of the middle is mobile element. genomic rd deletions defined lineages corresponding to the major spoligotype families beijing, eai, and haarlem, consistent with loci miru-vntr profiles. other controls consisted of ngs reads and complete whole genome sequences from genbank for intact-dr mtbc and for deleted-dr mtbc strain recognized as cas by its defining rd deletion. although many negative spoligotype strains are recorded as spoligotype-international-type in the sitvit international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least different loci miru-vntr and rd deletion families. mycobacterium tuberculosis complex mtbcnext generation sequencing ngscrispr-casspacerscas1is6110deleted-direct repeat region deleted-drregion of difference deletion rd deletionspoligotypemiru-vntrissue-copyright-statement© the author 2017 as positive controls, ngs was performed for three intact-dr isolates belonging to t3_eth, the largest multiple-drug-resistant -containing african-origin cluster in israel. crispr and crispr-flanking genomic regions are important for molecular epidemiology of mycobacterium tuberculosis complex strains, and potentially for adaptive immunity to phage and plasmid dna, and endogenous roles in the bacterium. analysis of ngs reads, and analysis of contigs obtained by manual pcr confirmed that all gold standard dr/crispr spacers were missing in the deleted-dr genomes. clonally specific deletion of crispr-flanking genes also was observed, including deletion of at least cas and cas genes.
 crispr and crispr-flanking genomic regions are important for molecular epidemiology of mycobacterium tuberculosis complex strains, and potentially for adaptive immunity to phage and plasmid dna, and endogenous roles in the bacterium. genotyping in the israel national mycobacterium reference center tel-aviv of over mtbc strains from 2008– showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions. two isolates of each of three negative spoligotype mtbc were subjected to next generation sequencing. as positive controls, ngs was performed for three intact-dr isolates belonging to t3_eth, the largest multiple-drug-resistant -containing african-origin cluster in israel. other controls consisted of ngs reads and complete whole genome sequences from genbank for intact-dr mtbc and for deleted-dr mtbc strain recognized as cas by its defining rd deletion.  ngs reads from negative spoligotype mtbc mapped to reference h37rv nc_ suggested that the dr/crispr regions were completely deleted except for retention of the middle is mobile element. clonally specific deletion of crispr-flanking genes also was observed, including deletion of at least cas and cas genes. genomic rd deletions defined lineages corresponding to the major spoligotype families beijing, eai, and haarlem, consistent with loci miru-vntr profiles. analysis of ngs reads, and analysis of contigs obtained by manual pcr confirmed that all gold standard dr/crispr spacers were missing in the deleted-dr genomes.  although many negative spoligotype strains are recorded as spoligotype-international-type in the sitvit international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least different loci miru-vntr and rd deletion families. we report for the first time negative spoligotype-associated total loss of crispr region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least crispr-associated genes cas and cas since cas deleted e.coli shows increased sensitivity to dna damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-dr strains and beijing family strains as both lack the cas gene. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mycobacterium tuberculosis complex mtbcnext generation sequencing ngscrispr-casspacerscas1is6110deleted-direct repeat region deleted-drregion of difference deletion rd deletionspoligotypemiru-vntrissue-copyright-statement© the author 2017 