BACKGROUND
dna barcoding seeks to develop a comprehensive species-specific sequence library for all eukaryotes  <cit> .
the  <dig> bp mitochondrial cytochrome c oxidase  <dig>  dna barcode  <cit>  is easily sequenced and provides greater than 97% species-level specificity for birds  <cit> , mammals  <cit> , fishes  <cit> , and various arthropods  <cit> .
however, conventional dna barcoding encounters two problems.
first, dna degradation in archival specimens and processed biological material  often prevents the recovery of pcr fragments longer than  <dig> bp, impeding barcode recovery  <cit> .
second, current approaches cannot be used for comprehensive analysis of environmental samples because high sequence variability necessitates the use of distinct primer sets for each major taxonomic group.
in this study, we propose the use of a "mini-barcode" sequence to overcome these problems.
we begin by identifying the minimum amount of sequence information required for accurate species identification.
we then test the gain in amplification success for smaller fragments in specimens with degraded dna.
finally, by targeting conserved priming sites within the barcode region we develop primers with the universality required for the analysis of all major eukaryotes.
RESULTS
to determine how much sequence information is required for identifications, we retrieved all co <dig> barcode sequences from genbank and calculated the probability of having species-specific barcodes for varied size fragments .
our analysis shows that while full-length dna barcodes perform best , 90% identification success is obtained with  <dig> bp regions and 95% success with  <dig> bp barcodes.
in another words, in 90% of the species tested a dna barcode of only  <dig> bp contains nucleotide substitution specific to members of a particular species.
having established the potential of mini-barcodes to identify unknown specimens, we proceeded to design primers and test their performance.
we designed universal primers for amplifying mini-barcodes by aligning co <dig> sequences from all major eukaryote groups  and identifying conserved amino acid strings for primers in the size range 120– <dig> bp.
we selected a single primer set for a  <dig> bp amplicon .
we tested the ability of this universal primer set to amplify dna extracts from  <dig>  specimens derived from  <dig> species of mammals, fishes, birds, and insects .
moreover, we examined another  <dig> dna extracts from plants, fungi, and macroalgae .
we obtained pcr amplicons from 92% of these species  and compared this success to that obtained via routine barcoding.
we were able to amplify mini-barcodes with greater success than full-length sequences in all groups but one  .
we verified that the  <dig> bp amplicons were co <dig> by sequencing approximately half of them .
one important application of mini-barcodes lies in obtaining sequence information from old type specimens.
we tested this approach on a collection of coleophora  which are difficult to identify because of their small size and cryptic morphology.
we successfully sequenced all  <dig> dried museum specimens collected from  <dig> to  <dig>  in one pcr pass with the universal mini-barcode primers and compared these specimens to  <dig> full-length barcodes from recently collected specimens of this genus .
comparison of sequence information obtained from these old specimens with recently collected samples provided excellent corroboration and species-level resolution.
mini-barcode sequences from old museum specimens formed monophyletic groups, containing zero or very low sequence divergence, with freshly collected specimens of the corresponding species .
CONCLUSIONS
the mini-barcode system dramatically broadens the applications of dna barcoding.
we have now demonstrated that sequence information can be reliably obtained from archival specimens or those with degraded dna.
further, the universality of the primers enables the recovery of comprehensive barcode information from environmental mixtures.
finally, the short universally-primed amplicon is ideal for sequence characterization through new parallelized high-throughput sequencing technologies, allowing inexpensive but comprehensive studies of biodiversity to be a realistic goal.

 dna barcoding seeks to develop a comprehensive species-specific sequence library for all eukaryotes. the bp mitochondrial cytochrome c oxidase dna barcode is easily sequenced and provides greater than 97% species-level specificity for birds, mammals, fishes, and various arthropods. however, conventional dna barcoding encounters two problems. first, dna degradation in archival specimens and processed biological material often prevents the recovery of pcr fragments longer than bp, impeding barcode recovery. second, current approaches cannot be used for comprehensive analysis of environmental samples because high sequence variability necessitates the use of distinct primer sets for each major taxonomic group. in this study, we propose the use of a "mini-barcode" sequence to overcome these problems. we begin by identifying the minimum amount of sequence information required for accurate species identification. we then test the gain in amplification success for smaller fragments in specimens with degraded dna. finally, by targeting conserved priming sites within the barcode region we develop primers with the universality required for the analysis of all major eukaryotes.  to determine how much sequence information is required for identifications, we retrieved all co barcode sequences from genbank and calculated the probability of having species-specific barcodes for varied size fragments. our analysis shows that while full-length dna barcodes perform best, 90% identification success is obtained with bp regions and 95% success with bp barcodes. in another words, in 90% of the species tested a dna barcode of only bp contains nucleotide substitution specific to members of a particular species. having established the potential of mini-barcodes to identify unknown specimens, we proceeded to design primers and test their performance. we designed universal primers for amplifying mini-barcodes by aligning co sequences from all major eukaryote groups and identifying conserved amino acid strings for primers in the size range 120– bp. we selected a single primer set for a bp amplicon. we tested the ability of this universal primer set to amplify dna extracts from specimens derived from species of mammals, fishes, birds, and insects. moreover, we examined another dna extracts from plants, fungi, and macroalgae. we obtained pcr amplicons from 92% of these species and compared this success to that obtained via routine barcoding. we were able to amplify mini-barcodes with greater success than full-length sequences in all groups but one. we verified that the bp amplicons were co by sequencing approximately half of them. one important application of mini-barcodes lies in obtaining sequence information from old type specimens. we tested this approach on a collection of coleophora which are difficult to identify because of their small size and cryptic morphology. we successfully sequenced all dried museum specimens collected from to in one pcr pass with the universal mini-barcode primers and compared these specimens to full-length barcodes from recently collected specimens of this genus. comparison of sequence information obtained from these old specimens with recently collected samples provided excellent corroboration and species-level resolution. mini-barcode sequences from old museum specimens formed monophyletic groups, containing zero or very low sequence divergence, with freshly collected specimens of the corresponding species.  the mini-barcode system dramatically broadens the applications of dna barcoding. we have now demonstrated that sequence information can be reliably obtained from archival specimens or those with degraded dna. further, the universality of the primers enables the recovery of comprehensive barcode information from environmental mixtures. finally, the short universally-primed amplicon is ideal for sequence characterization through new parallelized high-throughput sequencing technologies, allowing inexpensive but comprehensive studies of biodiversity to be a realistic goal. 
it may be difficult in practice, however, to retrieve a bp fragment from archival specimens, or from environmental samples. we used a bioinformatics analysis using all co barcode sequences from genbank and calculated the probability of having species-specific barcodes for varied size fragments. we further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens. we establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. we then developed a universal primer set for the amplification of mini-barcodes. this analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. in this study we address the important issue of minimum amount of sequence information required for identifying species in dna barcoding.
 the goal of dna barcoding is to develop a species-specific sequence library for all eukaryotes. a bp fragment of the cytochrome c oxidase gene has been used successfully for species-level identification in several animal groups. it may be difficult in practice, however, to retrieve a bp fragment from archival specimens, or from environmental samples.  we used a bioinformatics analysis using all co barcode sequences from genbank and calculated the probability of having species-specific barcodes for varied size fragments. this analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. we then developed a universal primer set for the amplification of mini-barcodes. we further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.  in this study we address the important issue of minimum amount of sequence information required for identifying species in dna barcoding. we establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. this approach will significantly broaden the application of dna barcoding in biodiversity studies. 