approximately 5,100 isolates of m. tuberculosis complex , predominately from patients in tb control programs in the northeast united states , are part of the wadsworth center spoligotype database . most of these strains have been collected through ongoing sentinel surveillance projects in massachusetts and new york city from 1996 to the present . in our strain collection ,
pcr amplifications were performed on extracted dna or cell suspensions , which were heat - killed at 80c for 1 hr in an oven .
spoligotype patterns were analyzed with bioimage whole band analysis v3.4 software ( genomic solutions , ann arbor , mi ) on a sun ultra10 workstation ( sun microsystems inc . ,
spoligotype patterns were given descriptive nomenclature according to the standard method ( 16 ) , along with a unique arbitrary numeric designation by the centers for disease control and prevention ( cdc ) .
is6110-based restriction fragment length polymorphism ( rflp ) analysis was performed , according to standard protocol ( 17 ) , at the wadsworth center ( albany , ny ) and the public health research institute ( new york city , ny ) , on approximately 3,700 of these isolates .
rflp patterns were analyzed with bioimage whole band analysis v3.4 software ( genomic solutions ) .
rflp pattern designations for sentinel surveillance isolates were assigned a unique arbitrary numerical designation by cdc .
sequence logo analysis was originally devised as a method to find blocks of related amino acids between protein sequences and display the information in an intuitive visual description that illustrates both the residue and the degree of conservation at each position ( 18 ) .
logo analysis has been used to look for functional and evolutionary relationships among groups of proteins and nucleic acids ( 19 ) .
spoligologo analysis ( 15 ) was accomplished by using weblogo software from the school of the biological sciences at the university of cambridge ( available from : url : http://www.bio.cam.ac.uk/seqlogo/ ) . to be compatible with weblogo ,
letter designations ( x = hybridization , o = no hybridization signal ) were used to denote the pattern of hybridization observed for each spoligotype pattern .
spoligotype patterns to be compared were entered directly into weblogo and a postscript file of the results was generated .
thus , we compared hybridization to spacer 1 in the group of selected spoligotypes , followed by analysis of spacer 2 , and so on for all 43 spacers . for convenience of illustration ,
two groups of isolates were chosen for spoligologo analysis . the first set consisted of 43 strains of m. bovis ( 13 ) in our collection .
the second set was 12 low - band ( exhibiting fewer than six copies of is6110 by rflp analysis ) m. tuberculosis isolates from vietnam - born , massachusetts sentinel surveillance case - patients .
vietnamese patients are the largest foreign - born group represented in the low - band data from massachusetts .
approximately 5,100 isolates of m. tuberculosis complex , predominately from patients in tb control programs in the northeast united states , are part of the wadsworth center spoligotype database . most of these strains have been collected through ongoing sentinel surveillance projects in massachusetts and new york city from 1996 to the present . in our strain collection ,
pcr amplifications were performed on extracted dna or cell suspensions , which were heat - killed at 80c for 1 hr in an oven .
spoligotype patterns were analyzed with bioimage whole band analysis v3.4 software ( genomic solutions , ann arbor , mi ) on a sun ultra10 workstation ( sun microsystems inc . , santa clara , ca ) .
spoligotype patterns were given descriptive nomenclature according to the standard method ( 16 ) , along with a unique arbitrary numeric designation by the centers for disease control and prevention ( cdc ) .
is6110-based restriction fragment length polymorphism ( rflp ) analysis was performed , according to standard protocol ( 17 ) , at the wadsworth center ( albany , ny ) and the public health research institute ( new york city , ny ) , on approximately 3,700 of these isolates .
rflp patterns were analyzed with bioimage whole band analysis v3.4 software ( genomic solutions ) .
rflp pattern designations for sentinel surveillance isolates were assigned a unique arbitrary numerical designation by cdc .
sequence logo analysis was originally devised as a method to find blocks of related amino acids between protein sequences and display the information in an intuitive visual description that illustrates both the residue and the degree of conservation at each position ( 18 ) .
logo analysis has been used to look for functional and evolutionary relationships among groups of proteins and nucleic acids ( 19 ) .
spoligologo analysis ( 15 ) was accomplished by using weblogo software from the school of the biological sciences at the university of cambridge ( available from : url : http://www.bio.cam.ac.uk/seqlogo/ ) . to be compatible with weblogo ,
letter designations ( x = hybridization , o = no hybridization signal ) were used to denote the pattern of hybridization observed for each spoligotype pattern .
spoligotype patterns to be compared were entered directly into weblogo and a postscript file of the results was generated .
thus , we compared hybridization to spacer 1 in the group of selected spoligotypes , followed by analysis of spacer 2 , and so on for all 43 spacers . for convenience of illustration ,
two groups of isolates were chosen for spoligologo analysis . the first set consisted of 43 strains of m. bovis ( 13 ) in our collection .
the second set was 12 low - band ( exhibiting fewer than six copies of is6110 by rflp analysis ) m. tuberculosis isolates from vietnam - born , massachusetts sentinel surveillance case - patients .
vietnamese patients are the largest foreign - born group represented in the low - band data from massachusetts .
figure 1 illustrates logo analysis with spoligotyping data . spoligotyping identified 28 different spoligotypes associated with m. bovis from 43 isolates in our collection .
letter designations were chosen for compatibility with the weblogo program as described in methods . the tallest x and o characters represent areas of absolute concordance between the patterns chosen for analysis .
where differences occur , the ratio of those spoligotypes showing hybridization to those that do not is represented by relative height differences of the characters in that column .
the resulting spoligologo ( figure 1b ) shows that spacers 20 , 25 , 26 , and 38 are present in all 28 spoligotype patterns .
the absence of spacers 3943 in these spoligotype patterns is consistent with an identification of m. bovis ( 13 ) .
the greatest polymorphism between the patterns appears in spacers 3 through 16 ( figure 1b ) .
spoligotype patterns probably evolve through the loss of spacer sequences through a variety of mechanisms ( 7,20 ) .
we try to extrapolate back to the hypothetical founder of these m. bovis isolates , which we believe had spacers 138 and not 3943 .
no isolate with this probable m. bovis founder spoligotype has yet been observed in the wadsworth center spoligotype database .
panel a illustrates the raw hybridization data followed by the same patterns coded for logo analysis . to be compatible with weblogo analysis , patterns were converted to a 43-character  long string consisting of the letters x and o. the letter x represents a positive hybridization , and o represents no hybridization detected for each of the 43 spacer sequences .
panel c shows the summary graphic of the spoligotypes by collapsing the data into a single row .
legend : x = hybridization observed to spacer , o= no hybridization observed to spacer , = positive hybridization in every spoligotype pattern for that individual spacer sequence , = no hybridization , =positive hybridization in > 50% of the patterns , = no hybridization in > 50% of the patterns .
foreign - born persons , especially those from regions with high tb case rates , are of concern for tb transmission in the united states ( 21,22 ) .
the low - band m. tuberculosis strains from vietnam - born patients , which we selected , produced another spoligologo pattern ( figure 2 ) .
the ability of spoligologo analysis to collapse even a small selection of spoligotype patterns from a select group of strains ( figure 2a ) into the possible founder spoligotype can be observed in the summary ( figure 2c ) .
however , additional typing methods would be required to verify that the strains are related , rather than exhibiting convergent evolution of their respective spoligotypes .
logo analysis on nine different spoligotypes observed for mycobacterium tuberculosis isolates from vietnam - born patients in massachusetts demonstrating fewer than six copies of is6110 by rflp analysis .
legend : x= hybridization observed to spacer , o= no hybridization observed to spacer , = positive hybridization in every spoligotype pattern for that individual spacer sequence , = no hybridization , =positive hybridization in > 50% of the patterns , = no hybridization in > 50% of the patterns .
as previously suggested ( 5,7,23 ) , m. tuberculosis strains that generally contain spacers 3343 may form a family that is an intermediate lineage between the beijing ( 11,12 ) and non - beijing m. tuberculosis strain families , such as haarlem ( 2 ) .
spoligotyping , microarrays , and dna - chips are all examples of reverse - hybridization array - based assays . although microarrays and dna - chips can contain thousands of bits of data , the principle behind them is similar to that of the spoligotype assay , which uses a simple 1 x 43 array .
array - based assays use reverse hybridization in which a labeled sample is probed against a series of proteins or nucleic acids that are bound to a solid support , such as a nylon membrane or silica . the result for each potential binding event
the binary nature of array - based assays allows the data to be analyzed usefully with algorithms associated with motif recognition , such as sequence logo analysis .
the relative low cost and simplicity of the spoligotype assay means it can be performed by many laboratories and the digital nature of the data facilitates the exchange of information among researchers .
the growth in the availability of array - based assays has outpaced the ability of conventional software analysis packages to provide every possible method of analysis .
customized versions of software are extremely expensive , and researchers , who want to implement these protocols without specialized software , lack methods of collating the large amounts of data .
problems arise when attempts are made to judge the significance of similar but nonidentical array data .
identifying possible families in these array patterns may be important in understanding the evolution and spread of pathogens such as m. tuberculosis .
our method for collapsing array - based data can be used to find and present patterns or signature motifs in these types of data . as previously noted ( 1 ) ,
cluster analysis of large rflp databases is difficult for a combination of reasons , including software failure and intralaboratory variations .
digital data , such as spoligotyping , mycobacterial interspersed repetitive unit ( 24 ) , and variable number tandem repeat analyses ( 25 ) , will probably form the basis for any large dna fingerprinting projects in the future ( 26 ) .
strains that cluster by one typing method must be analyzed by other methods to ensure that the groupings represent clusters of true relatedness and not cases of convergent evolution .
bioinformatic analyses , like logo analysis , may prove useful in obtaining further data from the large m. tuberculosis complex dna typing databases already in existence .
spoligologo analysis is a graphic method of presenting similar spoligotypes that may elicit useful insights into the geographic spread of tuberculosis .
potential families of tb strains could be identified on the basis of their logo ; these strains could then be analyzed by additional dna - typing methods to confirm the relationship , followed examining relevant patient data ( e.g. , country of birth ) .
a similar analysis was performed for the m. tuberculosis w - beijing family ( 11 ) that helped elucidate the evolution of a multidrug  resistant strain in new york city .
spoligologo analysis could help identify more of these families , determine their global origin , and evaluate their spread .
determining the sources and spread of tuberculosis is an important tool in preventing further infections .
understanding the geographic origin of an m. tuberculosis dna fingerprint could be useful , especially in understanding the sources and spread of strains in the u.s .
foreign - born population , among whom differentiating recently transmitted disease from reactivation of a past exposure can be difficult .
 spacer oligonucleotide ( spoligotyping ) analysis is a rapid polymerase chain reaction  based method of dna fingerprinting the mycobacterium tuberculosis complex . 
 we examined spoligotype data using a bioinformatic tool ( sequence logo analysis ) to elucidate undisclosed phylogenetic relationships and gain insights into the global dissemination of strains of tuberculosis . 
 logo analysis of spoligotyping data provides a simple way to describe a fingerprint signature and may be useful in categorizing unique spoligotypes patterns as they are discovered . 
 large databases of dna fingerprint information , such as those from the u.s . national tuberculosis genotyping and 
 surveillance network and the european concerted action on tuberculosis , contain information on thousands of strains from diverse regions . 
 the description of related spoligotypes has depended on exhaustive listings of the individual spoligotyping patterns . 
 logo analysis may become another useful graphic method of visualizing and presenting spoligotyping clusters from these databases . 
Methods
Isolates
DNA Fingerprinting Analyses
Spoligologo Analysis
Results
Discussion