supplementary material is available for this article at http://dx.doi.org/10.1007/s00414-006-0124-8 and is accessible for authorized users .
y - strs have key roles in the fields of forensic genetics , anthropological genetics and genealogy because of their ability to discriminate between male lineages and provide information about the relationships between them [ 1 , 2 ] . the y chromosome haplotype reference database  
provides a widely used compilation of haplotype information constructed from a  minimal haplotype  of nine loci or a  minht  +  swgdam core set  of 11 loci ( http://www.yhrd.org/index.html ) . some applications , however , require more y - strs .
for example , a study of 1,000 men from east asia found that almost 3% ( 27/1,003 ) shared the same 16-str haplotype   and thus would not be distinguished by standard analyses .
most of the strs on the y chromosome have now been identified , and a set of 52 was highlighted that seemed particularly useful because their unit size was 3 , they were single - copy , had a simple structure and showed variation in a set of eight diverse men .
these additional loci proved to be useful in the east asian study where 46 of them allowed a male lineage characteristic of the qing dynasty to be defined , but they clearly varied considerably in their diversity [ 4 , 5 ] and may vary in other properties that affect their usefulness as well .
in addition , it may often be impractical or impossible to type such a large number of markers .
us population data for 16 of them have been presented , but data from other loci and populations are lacking .
we have therefore established multiplex typing procedures for all of them and examined their variation in the y chromosome consortium ( ycc ) worldwide panel of men .
the ycc panel consists of 74 male and two female dnas ; the men may be broken down into 26 from africa , 26 from asia and the americas and 22 from europe or the middle east .
in addition , the haplogroup r individual previously typed with all of the new markers   was included in this study to facilitate consistent allele calling .
dna was amplified before use with the genomiphi whole genome amplification kit ( amersham biosciences , amersham , uk ) according to the manufacturer s recommendations .
a total of 52 polymorphic simple single - copy y - strs   were included in the present study .
the published primers had been designed to operate under a common set of conditions and were therefore used in this study , except that a g was added to the 5 end of the unlabelled primer if it was not already present to facilitate non - templated addition of an a to the labelled product strand .
loci were tested in silico for potential interactions between primers using the autodimer software , and suitable sets were assembled into small multiplexes for experimental assessment resulting in 16 multiplexes each consisting of 24 loci ( table  s1 ) .
polymerase chain reactions ( pcrs ) were set up in 20  l volumes containing 1 pcr buffer ( invitrogen , paisley , uk ) , 1.75  mm mgcl2 , 200  m deoxynucleotide triphosphates ( dntps ; amersham biosciences ) , 1.0 unit of platinum taq dna polymerase ( 5  u/l , invitrogen ) with 10  pg2  ng whole - genome - amplified dna and primer pairs at the concentrations shown in table  s1 .
thermal cycling was carried out in an mj research ( genetic research instrumentation , braintree , uk ) dna engine tetrad 2 starting with denaturation at 95c for 15  min , followed by 20 cycles of touchdown pcr : 94c for 30  s , 70c for 45  s , 72c for 1  min , with a 1c decrease in annealing temperature every cycle and then 15 cycles of standard pcr ( 94c for 30  s , 50c for 45  s , 72c for 1  min ) and finishing with extension at 60c for 45  min and storage at 4c .
products were analysed by mixing 1  l of pcr product with 15  l hi  di formamide and 0.2  l size marker ( cxr 60400 bases , promega uk , southampton , uk ) and running on 36  cm    50  m capillaries containing pop-4 polymer ( applied biosystems ) on an abi prism 3100 genetic analyzer ( applied biosystems , warrington , uk ) .
electrophoresis was carried out at 3  kv for 3  s followed by 15  kv for 45  min with a run temperature of 60c .
most loci were sequenced because of the lack of previous sequence data , to confirm previous results or to investigate the structure of intermediate - sized sizes .
such alleles were amplified using unlabelled primers and sequenced by the wellcome trust sanger institute small - scale sequencing facility using standard methods .
the 52 y - strs were examined in the 76 ycc samples and haplogroup r control individual , but the analyses presented in this paper ( tables  s2 , tables  s3 ) are based only on the ycc data to facilitate comparisons with other ycc results . as expected ,
no specific products were obtained from the two female ycc samples in the size range examined , and single peaks were seen in all males for 40 of the strs .
products from four loci were missing in one ( dys525 , dys589 , dys636 ) or two ( dys556 ) individuals .
these findings were reproducible and occurred in multiplex reactions that successfully amplified other loci , so that they may represent null alleles , but their structural basis remains to be determined , and they were treated conservatively as missing data in our analyses . 

table  1loci showing multiple peaks , missing peaks or intermediate alleleslocusintermediate allelecommentsdyf386s1two peaks in many individuals , excluded from analysisdyf390s1two peaks in many individuals , excluded from analysisdys448two peaks in two individualsdys52210 ( u2ains)intermediate - sized allele .
this insertion converts the reference sequence , which can be written atag atg ( atag)10 into atag at  a  g ( atag)10 , which therefore has 12 copies of the atag repeat , but differs from the regular allele 12dys525no product in one individual , two peaks in one individualdys53111 ( d6tins)intermediate - sized alleledys549two peaks in one individualdys556no product in two individualsdys567two peaks in two individualsdys576two peaks in two individualsdys589no product in one individualdys636no product in one individualu upstream , d downstream , ins insertionnomenclature based on the standard recommendations   loci showing multiple peaks , missing peaks or intermediate alleles u upstream , d downstream , ins insertion nomenclature based on the standard recommendations   two peaks were observed in many individuals for dyf390s1 and dyf386s1 , and we interpreted these as duplicated loci that happened to have the same sized alleles in the small number of individuals examined before ; these two strs were excluded from subsequent analyses .
five loci also showed two peaks of similar height in one ( dys525 , dys549 ) or two ( dys488 , dys567 , dys576 ) individuals , which may reflect rare duplications or somatic mutations in the ycc cell lines .
in addition , two loci showed fragment sizes that did not fall into the expected size classes : dys522 in one individual and dys531 in 11 individuals corresponding precisely to haplogroup q   and thus representing a variant characteristic of this haplogroup .
the structural basis of these variants was determined by sequencing and found to arise from insertion events in the flanking sequences between the strs and the primers ( table  1 ) .
null alleles , occasional duplications and intermediate alleles have been found in the standard y - strs , and so we concluded that 50 of the 52 new y - strs merited further consideration as loci for wider use .
the number of alleles ranged from two to 11 , the diversity from 0.05 to 0.90 and the variance from 0.04 to 7.89 ( table  2 ) .
all of these characteristics were correlated , probably because of their common dependence on the repeat count . to interpret the values
obtained , we have compared them with published data on the standard single - copy loci in the ycc panel . of the new loci ,
four ( dys481 , dys570 , dys576 and dys643 ) showed higher diversity than the most variable standard locus dys390 ( diversity  =  0.79 ) and 15 showed higher diversity than dys393 ( diversity  =  0.66 ; table  2 ) . the discrimination of haplotypes that are not distinguished by the commonly used markers is a particularly useful property .
as reported , eight pairs of ycc individuals carry haplotypes that are identical when the standard minimal set of y - strs is used .
two of these are from different populations ( mbuti pygmy / bantu speaker ; english / german ) and these were distinguished by seven and nine of the new loci , respectively .
the other six pairs are from isolated populations , and these were distinguished by 2 , 1 , 1 , 0 , 0 and 0 , respectively , of the new markers ( table  s4 ) . although a total of 15 loci contribute to this increased discrimination , all of the five distinguishable haplotypes could be separated using just two of the most variable loci , dys570 and dys576 . 

table  2variation of 50 new y - str loci in the ycc panellocusmean repeat countnumber of allelesdiversityvariancedys48123.3110.907.87dys57017.6110.863.89dys57617.370.822.16dys64311.190.822.33dys48515.970.781.92dyf406s110.670.751.26dys52211.260.741.03dys58912.360.731.07dys53311.260.721.03dys54912.150.720.92dys50512.160.711.00dys50811.480.711.44dys5259.970.710.95dys53111.040.690.39dys55611.760.671.03dys57210.350.640.61dys56511.560.640.71dys5949.570.630.87dys54011.440.620.52dys48713.260.621.38dys51110.950.620.73dys5739.940.620.73dys61712.450.621.06dys49515.350.610.76dys56710.460.610.72dys49714.240.600.51dys48813.460.580.91dys49211.650.560.45dys49012.970.562.43dys56811.170.560.92dys63611.350.560.49dys53710.940.530.43dys61811.840.510.37dys63810.940.490.34dys49112.150.460.40dys5788.140.410.40dys64011.230.390.39dys47611.240.360.29dys4949.040.350.28dys6419.640.330.93dys5549.050.310.36dys57510.040.250.22dys5838.030.220.12dys5907.930.220.23dys4807.930.220.16dys56911.030.180.11dys5309.120.170.09dys5809.140.110.19dys4728.020.080.04dys5799.020.050.89loci are ordered according to their diversity .
variation of 50 new y - str loci in the ycc panel loci are ordered according to their diversity .
we have investigated the properties of 52 new y - strs in a diverse worldwide set of males .
we found that two of the y - strs were multicopy and thus not well suited to some applications and that the remaining 50 loci differed substantially in their properties .
our measurements of allele numbers , diversity and variance were overall consistent with the previous report ; correlation coefficients ( r values ) were 0.47 , 0.58 and 0.67 , respectively , but differed for some individual loci .
the most variable y - str , in all respects , was dys481 , and this was not previously considered in detail because sequence data were not available before .
several other loci ( e.g. , dys570 , dys576 and dys643 ) may be particularly useful for increasing discrimination in forensic work , and the simple structure and mutational properties of this set make them the markers of choice for many population genetic studies .
this is illustrated by considering the correlation between mean repeat count and variance in repeat number of the 50 simple loci : it was far higher ( r  =  0.67 ) than the value reported for complex y - strs ( r  =  0.34 , ) , suggesting that the simple strs have simpler mutational mechanisms and may lead to more precise dates of lineages .
the data in table  2 and table  s3 now provide a basis for choosing the best simple loci and assembling them into a high - level multiplex reaction for more extensive population screening .
. 
  multiplex organization and primer concentrations ( doc 102 kb ) pcr product size range and allele range ( doc 92 kb ) haplotypes of the ycc dnas ( xls 58 kb ) ( note : this table is provided as an excel file and is the table mentioned in ce7 .
we transformed it into text because some journals insist on this , but the text version is difficult to interpret as ce5 highlights .
the loci are ordered according to their positions in multiplexes 116 in   , and it seems most consistent to keep the same order for   subdivision of minimal haplotypes by new y - strs ( doc 87 kb )
 we have established 16 small multiplex reactions of two  four loci to amplify 52 recently described single - copy simple y - strs and typed these loci in a worldwide panel of 74 diverse men and two women . 
 two y - strs were found to be commonly multicopy in this sample set and were excluded from the study . of the remaining 50 , four ( dys481 , dys570 , dys576 and dys643 ) showed higher diversities than the commonly used loci and 
 can potentially provide increased haplotype discrimination in both forensic and anthropological work . 
 ten loci showed occasional missing alleles , duplicated peaks or intermediate - sized alleles.electronic supplementary materialsupplementary material is available for this article at http://dx.doi.org/10.1007/s00414-006-0124-8 and is accessible for authorized users . 
Electronic supplementary material
Introduction
Materials and methods
Results
Discussion
Electronic supplementary material