BACKGROUND
the species of the mycobacterium tuberculosis complex  show a  <dig> % of similarity in their nucleotide sequence and their 16srrna do not differ between members, only m. canetti does  <cit> .
despite this identity in their genomes, a large number of long sequence polymorphisms , a variation in repetitive elements in the genome, and single nucleotide polymorphisms  have been detected  <cit> .
it is the diversity of such polymorphisms, which is taken for phylogenetic studies with clinical isolates.
in  <dig>  sreevatsan et al.
based on the presence of two snps in gyra <dig> and katg <dig>  classified all mtc isolates into three principal genetic groups or pggs  <cit> .
afterwards, brudey et al.
based on the “direct repeat” locus  diversity detected by spoligotyping, classified thousands of mtc clinical strains isolated worldwide in different lineages or families  <cit> .
these families were named according with their main geographical origin; latin american-mediterranean family  isolates, which are the cause of 15% of the new tb  cases detected each year worldwide, are highly prevalent in latin america and the mediterranean area  <cit> .
within this family a sub-lineage has been characterized by a genomic deletion known as rdrio, which was firstly detected in brazil, but it was widely spread throughout the world  <cit> .
haarlem family is ubiquitous throughout the world and accounts for 25% of the isolates extracted in europe, central america and the caribbean  <cit> .
the t family is an “ill defined” family that was characterized by default.
it includes over  <dig> shared international types  and it has been divided into  <dig> subgroups, from t <dig> to t <dig>  <cit> .
beijing family has become significant due to several multidrug-resistant  outbreaks identified  <cit> .
s family was identified predominantly in patients of italian origin  <cit> .
“x” family was described to be highly prevalent in north america  and central america , although some researchers correlate it with african-americans  <cit> .
central asian family  has been identified mostly in india, where presents a common sub-lineage called cas- <dig>  <cit> .
east african indonesian family  has a higher prevalence in southeast asia, particularly in the philippines, malaysia, vietnam and thailand  <cit> .
finally, the u family  does not meet the criteria of the other described families and it is considered separately  <cit> .
furthermore, a set of snps has been published as markers with phylogenetic value.
thus, seven phylogenetically different snp cluster groups  with  <dig> subgroups have been defined based on a set of snps, which have been related to the previously defined families  <cit> .
other significant polymorphisms were described as markers for particular families.
by way of illustration, snp in ag85c <dig> has been associated with lam family strains  <cit>  and among these strains a genomic deletion known as rdrio has been defined  <cit> .
likewise, some specific polymorphisms in ogt
44
, ung501
501
and mgtc
182
could serve as genetic markers for haarlem family  <cit> .
finally, a global phylogeny for m. tuberculosis was described based on lsps by six phylogeographical lineages, besides the m. bovis and m. canetti branches  <cit> , showing the prevalence of one of the lineages in europe and america, the euro-american lineage, which regroups the strains that had generally been described as principal genetic groups   <dig> and  <dig>  <cit> .
since  <dig> the genotyping of all clinical isolates of m. tuberculosis complex by is6110-based restriction fragment length polymorphism  and spoligotyping in aragon is systematically performed.
aragon is a region in the northeast of spain with  <dig> , <dig> registered inhabitants in the studied year  <dig> .
the aim of this study was to classify our collection of isolates into scg lineages, especially those belonging to “u”, “ill-defined” t families and isolates with no family associated.
with this intention, we have designed a method based on snps detection by multiplex-pcr and pyrosequencing  <cit> .
methods
sample selection
a total of  <dig> clinical isolates of m. tuberculosis complex collected as part of standard patient care from different areas within aragon in  <dig> had been previously identified, susceptibility to first line drugs tested and genotyped by using is6110-rflp and spoligotyping techniques.
these isolates had been assigned to a lineage or family after have been compared their spoligopatterns with those of the spoldb <dig>   <cit> , in the context of the surveillance network monitoring the potential transmission of tuberculosis in aragon.
for the scg determination assay  <dig> out of  <dig> were selected according to the following conditions: only one sample for each rflp-is <dig> cluster and the samples with a unique rflp.
once we confirmed that the isolates with the same spoligopattern were included in the same scg, a sample selection was made by choosing one isolate for each spoligopattern, resulting in  <dig> different isolates for further analysis .
reference strain h37rv was included as a control in each test performed.
<dig>
the analysis of the dr region was done in one case in which no positive hybridisation was obtained by spoligotyping using primers dr22-r  and dr43-f .
as no amplification was obtained a deletion of the region in this strain was considered and remains under study.
this isolate was considered in the study among the no sit assigned.
analysis of pggs and scgs and specific lineage polymorphisms
for the pyrosequencing assay nine snps that defined the seven scgs, were selected from the literature  <cit> : g.1977a > g, g.74092c > t, g.105139c > a, g.232574g > t, g.311613g > t, g.913274c > g, g.2460626c > a, g.3352929c > g, and gyra95g→c .
the snps presented in mgtc <dig>  in katg <dig> and in ag85c <dig> were identified by sequencing or pcr-rflp as previously described  <cit> .
rdrio deletion was detected by performing a multiplex-pcr  <cit> .
the pattern obtained for the gyra <dig> and katg <dig> polymorphisms was coupled to classify each isolate into the different pggs.
table adapted from bouakaze and co-workers  <cit>  and ainferred from filliol and coworkers  <cit> .
*new pattern scg-6c.
pyrosequencing analysis designed for snp detection
four multiplex pcr and one simplex pcr were developed to analyse the presence of the nine snps within our strains .
the snps location and gene sequence in h37rv genome were downloaded from the tuberculist website .
primers were designed using the qiagen® psq assay design v <dig>  software.
the programme provided the most suitable primers for dna amplification, labelling and pyrosequencing, as well as the optimal primer combination in multiplex pcrs .
for pyrosequencing, an indirect labelling protocol adapted from the literature was followed  <cit> .
first, the pcrs were performed using a universal biotinylated m <dig> primer and the specific couple of primers  for each snp.
in a second step, we used the pcr products to pyrosequence them with the subsequent sequencing primer.
each pcr mix contained: 16 mm 2so <dig>  67 mm tris–hcl ph <dig> ,  <dig> % tween- <dig>   <dig>  mm mgcl <dig>  200 μm dntp’,  <dig> u superhot taq ,  <dig> pmol of the biotinylated universal m <dig> primer , 1 μl of each couple of primers  and 1 μl of dna sample and was adjusted to a final volume of 25 μl with hplc water.
primers that were not being labelled with biotin in the pcr and the universal m <dig> primer were used at a concentration of  <dig> pmol/μl;  <dig> fmol/μl was used for those having the m <dig> tail.
a  <dig> pmol/μl concentration was employed for all sequence primers.
amplification was performed in a veriti® 96-well thermal cycler  for 2 min at 94°c followed by  <dig> cycles of 15 sec at 94°c, 30 sec at 64°c and 30 sec at 72°c.
the amplified products were visualized in a  <dig> % agarose gel and were loaded together with a 100 bp molecular weight marker .
in pcr plates of  <dig> wells we mixed 40 μl of binding buffer  and 3 μl of streptavidin-coated sepharose  beads to the 25 μl of pcr product, and the solution was mixed at 22/23°c for 20–30 min at  <dig>  r.p.m.
in an eppendorf thermomixer®.
using the vacuum prep tool the biotinylated pcr products were picked up with the 96-filter-unit and consequently immobilized on the streptavidin-coated sepharose beads.
then, the non-biotinylated dna was removed by placing the filter unit in the denaturation solution for 5 s, thus generating ssdna for pyrosequencing.
after neutralisation, the vacuum was switched off and the beads containing the pcr product were transferred to a 96-well plate with  <dig> pmol of each sequencing primer in 40 μl annealing buffer .
the sample was transferred into a reaction plate  and incubated for 2 min at 80°c.
the volume of enzymes, substrate and nucleotides calculated by pyromark q <dig> id software was added to the psq  <dig> cartridge accordingly.
pyrosequencing and snp analysis were done using the psq™96ma system and its software .
b
b
b
b
agene name and snp location in m. tuberculosis h37rv genome map .
one gene is listed when snp location is situated in that gene and two genes are listed when snp is intergenic.
bpcr name, amplicon expected size, and primers used.
RESULTS
we analysed the mtc strain family distribution of  <dig> isolates collected in  <dig> from across aragon .
within this set and according with the spoligotyping analysis, the haarlem genotype was the most frequent genotype , followed by the t “ill defined” family , u  and lam .
other genotypes showing a defined sit  grouped in smaller groups.
those isolates showing a pattern with no sit assigned in the spoldb <dig> database corresponded to  <dig> %.
among the  <dig> isolates,  <dig> isolates were included in the t, u and no sit groups representing the  <dig> % of the isolates.
accepting those with the same rflp-is <dig> genotype as clone-related isolates and therefore belonging to the same family or lineage, only one isolate of each rflp-is <dig> genotype,  <dig> isolates, were analysed by pyrosequencing .
once tested for the presence of the nine snps, we could confirm that those isolates with the same spoligopattern held into the same scg.
for further analysis one isolate for each spoligopattern was selected resulting a sample of  <dig> different mtc strains.
seven of the  <dig> strains according with their snps in gyra and katg genes were found to belong to pgg- <dig>   <dig> were included in pgg- <dig> and  <dig> were grouped in pgg- <dig>  the strains in pgg- <dig> shared the snps for scg- <dig>  scg- <dig>  scg- <dig> and scg-3a.
the scg-3b, scg-3c and scg- <dig> met the feature for pgg- <dig>  finally, pgg- <dig> embraced the isolates in scg-6a and a new scg that from now on it will be mentioned as “scg-6c”.
the described scg-6b pattern was only observed for the isolate of h37rv used as a control.
the distribution of these results is drawn and shown in figure  <dig> and table  <dig>  the vast majority of the strains  were classified in  <dig> scgs: scg-3b, scg- <dig> and scg-6a, in order of relevance.
it should be noted that isolates in scg- <dig> and scg-6b were not represented in this study.
*reference strain h37rv.
**new scg subgroup reported.
regarding the spoligo-families detected , the unique isolates in our study belonging to afri_ <dig> and eai7_bgd <dig> families were grouped in scg- <dig>  the beijing strain corresponded to the scg- <dig> and the unique cas isolate was included in scg-3a.
the m. bovis-bcg and m. bovis isolates  were grouped into scg- <dig>  the fifteen cases known to belong to the haarlem family were grouped in scg-3b.
the  <dig> lam and also the two s family strains were classified in scg- <dig>  two cases belonging to the x family were included in scg-3c.
our results showed that the  <dig> strains previously classified by spoligotyping in the ill-defined t, u family or with no sit assigned, were distributed among scg-3b, scg- <dig>  scg- <dig>  scg6-a and scg-6c .
scg-3b included twelve isolates, nine of them were not assigned to any of the spoligo-families, one isolate belonged to t <dig> family , one isolate to t4_ceu <dig> family  and one isolate to u family .
furthermore, additional snp at codon  <dig> in mgtc gene specific to the haarlem family was studied in these strains.
the codon mgtc
<dig> was present in eight of these isolates, including the classified as sit  <dig>
scg- <dig> included eleven isolates of t <dig> , u  and u-lam <dig>  families and four isolates which did not have any sit assigned.
they were studied to settle on their lam family membership.
all of them except two  presented the lam specific snp in ag85c <dig>  in addition, we found that two among the isolates tested, or five considering all the lam strains, contained the rdrio deletion, which is a feature of a subgroup of the lam family strains.
scg-6a included a total of  <dig> isolates, which belonged to t <dig> , t <dig> , t <dig> , t5_mad <dig> , u  and  <dig> isolates with not sit assigned.
none of them had either the snp in ag85c <dig> or the snp in mgtc
182
.
this scg-6a included the isolate of the most representative cluster in  <dig>  ara <dig> , which gathered  <dig> clinical cases since  <dig>  <cit> .
finally, two unrelated and different isolates presented the same new pattern named scg-6c, which only differs from scg-6a in one snp .
the first isolate  was related with the outbreak ara <dig>  and the second isolate  had not been previously reported in our region.
neither contained the snp in ag85c <dig> nor the snp in mgtc <dig> feature for lam or haarlem families respectively.
discussion
the euro-american lineage was found to be the predominant lineage of the m. tuberculosis complex in europe  <cit> .
the mdr tb studies carried out in spain showed the euro-american as the more prevalent lineage  <cit> , and that a few lam and haarlem strains, which belong to this lineage, played a major role in the spread of mdr strains  <cit> .
according to this, the 90% of the tuberculosis strains analysed in this work belong to this lineage.
our work allowed to classify a collection of mtc strains previously analysed by spoligotyping and rflp in aragon in lineages as well as in scgs by the detection of the  <dig> snps that define the  <dig> scgs  <cit>  together with pcr identification of katg <dig>  ag85c <dig> and mgtc <dig> polymorphisms.
all these single polymorphisms as a whole have proved to be an effective complement for both spoligotyping and rflp techniques that enhance their sensibility, especially in those families identified at the beginning as t, u and orphan.
a notorious circumstance to remark in our population was that the two largest clusters of m. tuberculosis strains, named ara <dig> and ara <dig>  belonged to t and unclassified groups of families.
besides, ara <dig> had caused an outbreak since  <dig>  what resulted in around the 20% of cases of tuberculosis  <cit> .
this fact allows the classification of these strains into more resolved families.
in addition, the  <dig> snps detection by using a pyrosequencing assay leads to obtain quick and reliable results at an affordable cost  <cit> .
we have shown that some strains identified by spoligotyping as t, u or even orphan, which represent in our study the  <dig> % of the isolates, belong in fact to defined families that could be assigned by using the aforementioned polymorphism set.
in few occasions it was not possible to group those strains into a family with certainty, therefore snp detection in ag85c <dig> and mgtc <dig> was needed.
thus, regarding scg-3b, the most prevalent in our community, the addition of a specific snp detection as mgtc <dig>  a characteristic snp of the haarlem family, gave more specific information.
filliol and collaborators joined in this scg-3b basically haarlem isolates, but also some t, lam, and orphan strains  <cit> .
it either happened the same concerning scg- <dig>  the second most prevalent scg in aragon, in which filliol and collaborators included essentially lam strains, but also t, haarlem, s, unknown and orphan isolates  <cit> .
the pyrosequencing method applied allows to include an isolate in scg- <dig>  further the ag85c <dig> asserts of its lam membership even if spoligotyping had not been detected it at first.
regarding scg-6a, which was the third group of relevance in our study, we believe it includes the vast majority of the t isolates that would group as the “authentic t” isolates, being a more evolved strains since they belong to the pgg- <dig>  another achievement of this snps set has been the discovery of the two genetically and epidemiologically not linked isolates included in the new “scg-6c”.
it suggests that the tubercle bacillus is incessantly varying and highlights the value of snps to follow the evolution of m. tuberculosis complex.
concerning the pgg determination, around 70% of the strains circulating in our community grouped in the pgg- <dig>  this study provides a first inside into the structure of the m. tuberculosis population in aragon and spain.
the strains causing the largest clusters were classified as belonged to pgg- <dig>  ara <dig>  and ara <dig> , what means these modern strains are causing the more cases of tb in our region, both of them belong to the euro-american lineage  <cit> .
comparing our results with a study carried out in london  <cit> , we appreciate less diversity regarding spoligo-families probably due to the minor rate of patients that born abroad in respect to the london population.
they characterised the mtbc strains using snps, however some of the isolates remained unclassified.
a recent publication designed an algorithmic differentiating euro-american based on polymorphic snps in  <dig> genes in an extend collection of well-classified members of the mtb complex  <cit> .
however, the application of the analysis of the set of snps previously described  <cit>  selected in this study allowed us to assign  <dig> strains sharing different spoligotypes to different scgs and families in the mtc, specially those assigned to the ill defined t and other unclassified.
we believe that classifying our isolates in the precedent pggs previously described along with the scgs and spoligo-families provided the appropriate information to better understand the phylogenetic background of the aragonian strains being this approach applicable to other isolates of any geographical location.
CONCLUSIONS
in conclusion, the current study shows that the polymorphisms selected have been quite useful to complement and enrich the characterization of all isolates, specifically for those that would not have been classified by other routine techniques.
although more studies with a larger amount of samples would be required, this work has allowed us to do a better classification of aragonian strains into scgs and pggs by using pyrosequencing and conventional pcr, and in some cases, to assign strains to a certain lineage.
besides, the description of a new pattern shared by two isolates “scg-6c” reinforces the interest of snps to follow the evolution of m. tuberculosis complex.
in addition, our work describes the successful development of a multiplex-pcr and pyrosequencing assay based on snp detection as a purpose to classify m. tuberculosis isolates into more resolved phylogenetic groups called scgs and to determine the principal genetic groups.
therefore we suggest the use of this pyrosequencing technique as a complement to current phylogenetic and epidemiological investigations.
ethics statement
the ethical committee of the aragon government approved the study and the protocols for collecting the bacterial strains from patients.
any human sample was collected.
abbreviations
mtc: mycobacterium tuberculosis complex; lsps: long sequence polymorphisms; snps: single nucleotide polymorphisms; pgg: principal genetic group; dr: direct repeats; lam: latin american-mediterranean family; tb: tuberculosis; sit: shared international type; mdr: multidrug resistant; cas: central asian family; eai: east african indonesian family; scg: snp cluster groups; rflp: restriction fragment length polymorphism; spoldb4: fourth international spoligotyping database.
competing interests
none of the investigators has any financial interest or financial conflict with the subject matter or materials discussed in this report.
all authors read and approved the final manuscript.
authors’ contributions
ss and jd contributed to the study design, ac, ms design and the development of the pyrosequencing technique, cm, mji, mal, mav, mf facilitate the background and support the mycobacterial isolates genotyping studies.
ac and ss analysed data and drafted the manuscript.
all authors read and approved the final manuscript.

 the species of the mycobacterium tuberculosis complex show a % of similarity in their nucleotide sequence and their 16srrna do not differ between members, only m. canetti does. despite this identity in their genomes, a large number of long sequence polymorphisms, a variation in repetitive elements in the genome, and single nucleotide polymorphisms have been detected. it is the diversity of such polymorphisms, which is taken for phylogenetic studies with clinical isolates. in sreevatsan et al. based on the presence of two snps in gyra and katg classified all mtc isolates into three principal genetic groups or pggs. afterwards, brudey et al. based on the “direct repeat” locus diversity detected by spoligotyping, classified thousands of mtc clinical strains isolated worldwide in different lineages or families. these families were named according with their main geographical origin; latin american-mediterranean family isolates, which are the cause of 15% of the new tb cases detected each year worldwide, are highly prevalent in latin america and the mediterranean area. within this family a sub-lineage has been characterized by a genomic deletion known as rdrio, which was firstly detected in brazil, but it was widely spread throughout the world. haarlem family is ubiquitous throughout the world and accounts for 25% of the isolates extracted in europe, central america and the caribbean. the t family is an “ill defined” family that was characterized by default. it includes over shared international types and it has been divided into subgroups, from t to t. beijing family has become significant due to several multidrug-resistant outbreaks identified. s family was identified predominantly in patients of italian origin. “x” family was described to be highly prevalent in north america and central america, although some researchers correlate it with african-americans. central asian family has been identified mostly in india, where presents a common sub-lineage called cas-. east african indonesian family has a higher prevalence in southeast asia, particularly in the philippines, malaysia, vietnam and thailand. finally, the u family does not meet the criteria of the other described families and it is considered separately. furthermore, a set of snps has been published as markers with phylogenetic value. thus, seven phylogenetically different snp cluster groups with subgroups have been defined based on a set of snps, which have been related to the previously defined families. other significant polymorphisms were described as markers for particular families. by way of illustration, snp in ag85c has been associated with lam family strains and among these strains a genomic deletion known as rdrio has been defined. likewise, some specific polymorphisms in ogt 44, ung501 501 and mgtc 182 could serve as genetic markers for haarlem family. finally, a global phylogeny for m. tuberculosis was described based on lsps by six phylogeographical lineages, besides the m. bovis and m. canetti branches, showing the prevalence of one of the lineages in europe and america, the euro-american lineage, which regroups the strains that had generally been described as principal genetic groups and. since the genotyping of all clinical isolates of m. tuberculosis complex by is6110-based restriction fragment length polymorphism and spoligotyping in aragon is systematically performed. aragon is a region in the northeast of spain with, registered inhabitants in the studied year. the aim of this study was to classify our collection of isolates into scg lineages, especially those belonging to “u”, “ill-defined” t families and isolates with no family associated. with this intention, we have designed a method based on snps detection by multiplex-pcr and pyrosequencing. methods sample selection a total of clinical isolates of m. tuberculosis complex collected as part of standard patient care from different areas within aragon in had been previously identified, susceptibility to first line drugs tested and genotyped by using is6110-rflp and spoligotyping techniques. these isolates had been assigned to a lineage or family after have been compared their spoligopatterns with those of the spoldb, in the context of the surveillance network monitoring the potential transmission of tuberculosis in aragon. for the scg determination assay out of were selected according to the following conditions: only one sample for each rflp-is cluster and the samples with a unique rflp. once we confirmed that the isolates with the same spoligopattern were included in the same scg, a sample selection was made by choosing one isolate for each spoligopattern, resulting in different isolates for further analysis. reference strain h37rv was included as a control in each test performed. the analysis of the dr region was done in one case in which no positive hybridisation was obtained by spoligotyping using primers dr22-r and dr43-f. as no amplification was obtained a deletion of the region in this strain was considered and remains under study. this isolate was considered in the study among the no sit assigned. analysis of pggs and scgs and specific lineage polymorphisms for the pyrosequencing assay nine snps that defined the seven scgs, were selected from the literature: g.1977a > g, g.74092c > t, g.105139c > a, g.232574g > t, g.311613g > t, g.913274c > g, g.2460626c > a, g.3352929c > g, and gyra95g→c. the snps presented in mgtc in katg and in ag85c were identified by sequencing or pcr-rflp as previously described. rdrio deletion was detected by performing a multiplex-pcr. the pattern obtained for the gyra and katg polymorphisms was coupled to classify each isolate into the different pggs. table adapted from bouakaze and co-workers and ainferred from filliol and coworkers. *new pattern scg-6c. pyrosequencing analysis designed for snp detection four multiplex pcr and one simplex pcr were developed to analyse the presence of the nine snps within our strains. the snps location and gene sequence in h37rv genome were downloaded from the tuberculist website. primers were designed using the qiagen® psq assay design v software. the programme provided the most suitable primers for dna amplification, labelling and pyrosequencing, as well as the optimal primer combination in multiplex pcrs. for pyrosequencing, an indirect labelling protocol adapted from the literature was followed. first, the pcrs were performed using a universal biotinylated m primer and the specific couple of primers for each snp. in a second step, we used the pcr products to pyrosequence them with the subsequent sequencing primer. each pcr mix contained: 16 mm 2so 67 mm tris–hcl ph, % tween-  mm mgcl 200 μm dntp’, u superhot taq, pmol of the biotinylated universal m primer, 1 μl of each couple of primers and 1 μl of dna sample and was adjusted to a final volume of 25 μl with hplc water. primers that were not being labelled with biotin in the pcr and the universal m primer were used at a concentration of pmol/μl; fmol/μl was used for those having the m tail. a pmol/μl concentration was employed for all sequence primers. amplification was performed in a veriti® 96-well thermal cycler for 2 min at 94°c followed by cycles of 15 sec at 94°c, 30 sec at 64°c and 30 sec at 72°c. the amplified products were visualized in a % agarose gel and were loaded together with a 100 bp molecular weight marker. in pcr plates of wells we mixed 40 μl of binding buffer and 3 μl of streptavidin-coated sepharose beads to the 25 μl of pcr product, and the solution was mixed at 22/23°c for 20–30 min at r.p.m. in an eppendorf thermomixer®. using the vacuum prep tool the biotinylated pcr products were picked up with the 96-filter-unit and consequently immobilized on the streptavidin-coated sepharose beads. then, the non-biotinylated dna was removed by placing the filter unit in the denaturation solution for 5 s, thus generating ssdna for pyrosequencing. after neutralisation, the vacuum was switched off and the beads containing the pcr product were transferred to a 96-well plate with pmol of each sequencing primer in 40 μl annealing buffer. the sample was transferred into a reaction plate and incubated for 2 min at 80°c. the volume of enzymes, substrate and nucleotides calculated by pyromark q id software was added to the psq cartridge accordingly. pyrosequencing and snp analysis were done using the psq™96ma system and its software. b b b b agene name and snp location in m. tuberculosis h37rv genome map. one gene is listed when snp location is situated in that gene and two genes are listed when snp is intergenic. bpcr name, amplicon expected size, and primers used.  we analysed the mtc strain family distribution of isolates collected in from across aragon. within this set and according with the spoligotyping analysis, the haarlem genotype was the most frequent genotype, followed by the t “ill defined” family, u and lam. other genotypes showing a defined sit grouped in smaller groups. those isolates showing a pattern with no sit assigned in the spoldb database corresponded to %. among the isolates, isolates were included in the t, u and no sit groups representing the % of the isolates. accepting those with the same rflp-is genotype as clone-related isolates and therefore belonging to the same family or lineage, only one isolate of each rflp-is genotype, isolates, were analysed by pyrosequencing. once tested for the presence of the nine snps, we could confirm that those isolates with the same spoligopattern held into the same scg. for further analysis one isolate for each spoligopattern was selected resulting a sample of different mtc strains. seven of the strains according with their snps in gyra and katg genes were found to belong to pgg- were included in pgg- and were grouped in pgg- the strains in pgg- shared the snps for scg- scg- scg- and scg-3a. the scg-3b, scg-3c and scg- met the feature for pgg- finally, pgg- embraced the isolates in scg-6a and a new scg that from now on it will be mentioned as “scg-6c”. the described scg-6b pattern was only observed for the isolate of h37rv used as a control. the distribution of these results is drawn and shown in figure  and table  the vast majority of the strains were classified in scgs: scg-3b, scg- and scg-6a, in order of relevance. it should be noted that isolates in scg- and scg-6b were not represented in this study. *reference strain h37rv. **new scg subgroup reported. regarding the spoligo-families detected, the unique isolates in our study belonging to afri_ and eai7_bgd families were grouped in scg- the beijing strain corresponded to the scg- and the unique cas isolate was included in scg-3a. the m. bovis-bcg and m. bovis isolates were grouped into scg- the fifteen cases known to belong to the haarlem family were grouped in scg-3b. the lam and also the two s family strains were classified in scg- two cases belonging to the x family were included in scg-3c. our results showed that the strains previously classified by spoligotyping in the ill-defined t, u family or with no sit assigned, were distributed among scg-3b, scg- scg- scg6-a and scg-6c. scg-3b included twelve isolates, nine of them were not assigned to any of the spoligo-families, one isolate belonged to t family, one isolate to t4_ceu family and one isolate to u family. furthermore, additional snp at codon in mgtc gene specific to the haarlem family was studied in these strains. the codon mgtc was present in eight of these isolates, including the classified as sit scg- included eleven isolates of t, u and u-lam families and four isolates which did not have any sit assigned. they were studied to settle on their lam family membership. all of them except two presented the lam specific snp in ag85c in addition, we found that two among the isolates tested, or five considering all the lam strains, contained the rdrio deletion, which is a feature of a subgroup of the lam family strains. scg-6a included a total of isolates, which belonged to t, t, t, t5_mad, u and isolates with not sit assigned. none of them had either the snp in ag85c or the snp in mgtc 182. this scg-6a included the isolate of the most representative cluster in ara, which gathered clinical cases since. finally, two unrelated and different isolates presented the same new pattern named scg-6c, which only differs from scg-6a in one snp. the first isolate was related with the outbreak ara and the second isolate had not been previously reported in our region. neither contained the snp in ag85c nor the snp in mgtc feature for lam or haarlem families respectively. discussion the euro-american lineage was found to be the predominant lineage of the m. tuberculosis complex in europe. the mdr tb studies carried out in spain showed the euro-american as the more prevalent lineage, and that a few lam and haarlem strains, which belong to this lineage, played a major role in the spread of mdr strains. according to this, the 90% of the tuberculosis strains analysed in this work belong to this lineage. our work allowed to classify a collection of mtc strains previously analysed by spoligotyping and rflp in aragon in lineages as well as in scgs by the detection of the snps that define the scgs together with pcr identification of katg ag85c and mgtc polymorphisms. all these single polymorphisms as a whole have proved to be an effective complement for both spoligotyping and rflp techniques that enhance their sensibility, especially in those families identified at the beginning as t, u and orphan. a notorious circumstance to remark in our population was that the two largest clusters of m. tuberculosis strains, named ara and ara belonged to t and unclassified groups of families. besides, ara had caused an outbreak since what resulted in around the 20% of cases of tuberculosis. this fact allows the classification of these strains into more resolved families. in addition, the snps detection by using a pyrosequencing assay leads to obtain quick and reliable results at an affordable cost. we have shown that some strains identified by spoligotyping as t, u or even orphan, which represent in our study the % of the isolates, belong in fact to defined families that could be assigned by using the aforementioned polymorphism set. in few occasions it was not possible to group those strains into a family with certainty, therefore snp detection in ag85c and mgtc was needed. thus, regarding scg-3b, the most prevalent in our community, the addition of a specific snp detection as mgtc a characteristic snp of the haarlem family, gave more specific information. filliol and collaborators joined in this scg-3b basically haarlem isolates, but also some t, lam, and orphan strains. it either happened the same concerning scg- the second most prevalent scg in aragon, in which filliol and collaborators included essentially lam strains, but also t, haarlem, s, unknown and orphan isolates. the pyrosequencing method applied allows to include an isolate in scg- further the ag85c asserts of its lam membership even if spoligotyping had not been detected it at first. regarding scg-6a, which was the third group of relevance in our study, we believe it includes the vast majority of the t isolates that would group as the “authentic t” isolates, being a more evolved strains since they belong to the pgg- another achievement of this snps set has been the discovery of the two genetically and epidemiologically not linked isolates included in the new “scg-6c”. it suggests that the tubercle bacillus is incessantly varying and highlights the value of snps to follow the evolution of m. tuberculosis complex. concerning the pgg determination, around 70% of the strains circulating in our community grouped in the pgg- this study provides a first inside into the structure of the m. tuberculosis population in aragon and spain. the strains causing the largest clusters were classified as belonged to pgg- ara and ara, what means these modern strains are causing the more cases of tb in our region, both of them belong to the euro-american lineage. comparing our results with a study carried out in london, we appreciate less diversity regarding spoligo-families probably due to the minor rate of patients that born abroad in respect to the london population. they characterised the mtbc strains using snps, however some of the isolates remained unclassified. a recent publication designed an algorithmic differentiating euro-american based on polymorphic snps in genes in an extend collection of well-classified members of the mtb complex. however, the application of the analysis of the set of snps previously described selected in this study allowed us to assign strains sharing different spoligotypes to different scgs and families in the mtc, specially those assigned to the ill defined t and other unclassified. we believe that classifying our isolates in the precedent pggs previously described along with the scgs and spoligo-families provided the appropriate information to better understand the phylogenetic background of the aragonian strains being this approach applicable to other isolates of any geographical location.  in conclusion, the current study shows that the polymorphisms selected have been quite useful to complement and enrich the characterization of all isolates, specifically for those that would not have been classified by other routine techniques. although more studies with a larger amount of samples would be required, this work has allowed us to do a better classification of aragonian strains into scgs and pggs by using pyrosequencing and conventional pcr, and in some cases, to assign strains to a certain lineage. besides, the description of a new pattern shared by two isolates “scg-6c” reinforces the interest of snps to follow the evolution of m. tuberculosis complex. in addition, our work describes the successful development of a multiplex-pcr and pyrosequencing assay based on snp detection as a purpose to classify m. tuberculosis isolates into more resolved phylogenetic groups called scgs and to determine the principal genetic groups. therefore we suggest the use of this pyrosequencing technique as a complement to current phylogenetic and epidemiological investigations. ethics statement the ethical committee of the aragon government approved the study and the protocols for collecting the bacterial strains from patients. any human sample was collected. abbreviations mtc: mycobacterium tuberculosis complex; lsps: long sequence polymorphisms; snps: single nucleotide polymorphisms; pgg: principal genetic group; dr: direct repeats; lam: latin american-mediterranean family; tb: tuberculosis; sit: shared international type; mdr: multidrug resistant; cas: central asian family; eai: east african indonesian family; scg: snp cluster groups; rflp: restriction fragment length polymorphism; spoldb4: fourth international spoligotyping database. competing interests none of the investigators has any financial interest or financial conflict with the subject matter or materials discussed in this report. all authors read and approved the final manuscript. authors’ contributions ss and jd contributed to the study design, ac, ms design and the development of the pyrosequencing technique, cm, mji, mal, mav, mf facilitate the background and support the mycobacterial isolates genotyping studies. ac and ss analysed data and drafted the manuscript. all authors read and approved the final manuscript. 
different polymorphisms have been described as markers to classify the lineages of the mycobacterium tuberculosis complex. the analysis of nine single nucleotide polymorphisms was used to describe seven snps cluster groups. besides, the description of a new pattern shared by two isolates “scg-6c” reinforces the interest of snps to follow the evolution of m. tuberculosis complex. the m. tuberculosis complex isolates collected in in our region were analysed., ag85c and rdrio deletion were detected. this work has permitted to achieve a better classification of aragonian strains into scgs and in some cases, to assign strains to its certain lineage.
 different polymorphisms have been described as markers to classify the lineages of the mycobacterium tuberculosis complex. the analysis of nine single nucleotide polymorphisms was used to describe seven snps cluster groups. we attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing.  the m. tuberculosis complex isolates collected in in our region were analysed. a new method based on multiplex-pcrs and pyrosequencing to analyse these snps was designed. for the pyrosequencing assay nine snps that defined the seven scgs were selected from the literature: and gyra in addition, snps in katg mgtc 182, ag85c and rdrio deletion were detected.  this work has permitted to achieve a better classification of aragonian strains into scgs and in some cases, to assign strains to its certain lineage. besides, the description of a new pattern shared by two isolates “scg-6c” reinforces the interest of snps to follow the evolution of m. tuberculosis complex. m. tuberculosissnppyrosequencingscglineagescluster 