TECHNICAL NOTE

An efficient multiplex genotyping approach for detectingthe major worldwide human Y-chromosome haplogroups

Mannis van Oven & Arwin Ralf & Manfred Kayser

Received: 21 June 2011 /Accepted: 13 July 2011# The Author(s). This article is published with open access at Springerlink.com 2011

Abstract The Y chromosome is paternally inherited andtherefore serves as an evolutionary marker of patrilinealdescent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be repre-sented as a monophyletic phylogenetic tree in which thebranches (haplogroups) are defined by at least one SNP.Previous human population genetics research has produceda wealth of knowledge about the worldwide distribution ofY-SNP haplogroups. Here, we apply previous and veryrecent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplexgenotyping assays that allow for the hierarchical detectionof 28 Y-SNPs defining the major worldwide Y haplogroups.PCR amplicons were kept small to make the methodsensitive and thereby applicable to DNA of limited amountand/or quality such as in forensic settings. These Y-SNPassays thus form a valuable tool for researchers in the fieldsof forensic genetics and genetic anthropology to infer aman's patrilineal bio-geographic ancestry from DNA.

Keywords Y chromosome . Y-SNP. Haplogroup .

Patrilineal ancestry. Bio-geographic ancestry.MultiplexSNaPshot

Introduction

Knowledge about the bio-geographic ancestry revealed fromcrime-scene samples can be relevant for investigative intelli-

gence purposes in search for unknown sample donors whousually cannot be identified via conventional forensic STRprofiling. DNA-based bio-geographic ancestry inference isalso applied in genealogical and anthropological research forvarious purposes. The human Y chromosome is widelystudied as an evolutionary marker of patrilineal descent. Awell-established Y-chromosome phylogeny is available [6]and is continuously being expanded as novel SNPs arediscovered. Awealth of data has been produced previouslyon the worldwide distribution and allele frequencies ofnumerous Y-SNPs and the respective Y haplogroups theydefine. Here, we take advantage of existing knowledge onthe Y-SNP phylogeny and worldwide Y haplogroup distri-bution and introduce two Y-SNP multiplex assays, based onsingle-base primer extension (SNaPshot) technology, forthe detection of the major worldwide Y haplogoups.Together with well-known Y-SNPs, we have also includedsome relatively novel Y-SNPs such as M522 [4], M526 [4],P326 [8] and M412 [9], acknowledging most recent progressin Y-chromosome research.

Materials and methods

DNA samples

A subset of DNA samples from the HapMap 3 referencepanel [1], belonging to various Y haplogroups, wasobtained from the Coriell Institute for Medical Research(http://www.coriell.org/).

Primer design

Primers were designed using Primer3Plus [11] with a Tmaround 60C for PCR primers and around 55C for

M. van Oven (*) :A. Ralf :M. KayserDepartment of Forensic Molecular Biology,Erasmus MC University Medical Center Rotterdam,3000 CA Rotterdam, The Netherlandse-mail: m.vanoven@erasmusmc.nl

Int J Legal MedDOI 10.1007/s00414-011-0605-2

extension primers. Potential interactions between primers inthe same multiplex were evaluated with the AutoDimerversion 1.0 software [12]. In order to minimize allelicdropouts due to primer mismatches, we avoided as much aspossible that primer-annealing sites overlapped with knownY-chromosome polymorphisms. Extension primers werevaried in length through the addition of 5 non-homologous poly(GACT) tails to ensure electrophoreticseparation of extended fragments.

PCR amplification

Multiplex PCR amplification was carried out in a reactionvolume of 6 L, containing 1 GeneAmp PCR Gold Buffer(Applied Biosystems, CA, USA), 4.5 mM MgCl2 (AppliedBiosystems), 100 M of each dNTP (Roche, Mannheim,Germany), 0.35 units of AmpliTaq Gold DNA polymerase(Applied Biosystems), 12 ng of genomic DNA template,and PCR primers (desalted; Metabion, Martinsried,Germany) in concentrations as specified in Tables 1 and 2.The reactions were performed in a Dual 384-well GeneAmpPCR System 9700 (Applied Biosystems) using the followingcycling conditions: 10 min at 95C, followed by 30 cycles of94C for 15 s, 60C for 45 s, and a final extension at 60Cfor 5 min. PCR products were purified by adding 2 LExoSAP-IT (USB Corporation, OH, USA) to 6 L PCRproduct, followed by incubation at 37C for 30 min and 80Cfor 15 min.

Single-base extension

Multiplex single-base primer extension was carried out in areaction volume of 6 L, containing 1 L SNaPshotReady Reaction Mix (Applied Biosystems), 1 Lpurified PCR product, and extension primers (HPLC-purified; Metabion, Martinsried, Germany) in concen-trations as specified in Tables 1 and 2. The reactionswere performed in a Dual 384-well GeneAmp PCRSystem 9700 (Applied Biosystems) using the followingcycling conditions: 2 min at 96C, followed by 25 cyclesof 96C for 10 s, 50C for 5 s, and 60C for 30 s. Thereaction products were purified by adding 1 unit of ShrimpAlkaline Phosphatase (USB Corporation) to 6 L ofextension product, followed by incubation at 37C for45 min and 75C for 15 min.

Capillary electrophoresis

The extended fragments were separated and detected bycapillary electrophoresis on a 3130xl Genetic Analyzer(Applied Biosystems) using POP-7 polymer. A mixture of1 L purified extension product, 8.7 L Hi-Di formamide(Applied Biosystems) and 0.3 L GeneScan-120 LIZ

internal size standard (Applied Biosystems) was run with10 s injection time at 1.2 kVand 500 s run time at 15.0 kV.Results were analysed using GeneMapper version 3.7software (Applied Biosystems).

Results and discussion

Two genotyping multiplex assays were developed targetinga total of 28 Y-SNPs that define the major worldwide Y-chromosome haplogroups (Fig. 1). During the course ofthis work, a paper was published that reported a reorgani-zation of the deepest clades of the Y-chromosome phylog-eny, one of the consequences being that marker M91 nolonger defines a monophyletic haplogroup A, but rathershould be placed on the stem leading to the BCDEF(also referred to as BT) clade [5]. We have incorporatedthis change in our tables and figures to conform with thelatest Y-chromosome topology. Furthermore, we tookadvantage of some recently discovered Y-SNPs (P326,M526, M522 and M412) that, as far as we know, were notincluded in previous Y genotyping systems [e.g. 2, 3, 10].Of these novel SNPs, P326 (also known as L298) definesa new branch that joins haplogroups L and T into a singleclade now called LT [8]. M526 is located downstream ofmarker M9 and encompasses haplogroups K1 to K4 aswell as M to S [4]; the branch defined by M526 is nowreferred to as haplogroup K, and the former haplogroup K(defined by M9) is now relabelled as KLT. M522 (alsoknown as L16 or S138) defines a new node withinhaplogroup F that encompasses haplogroups I, J andKLT [4] and is referred to as haplogroup IJKLT. M412(also known as L51 or S167) defines a significantsubhaplogroup within haplogroup R that is most abundantin western parts of Europe [9].

The 28 Y-SNPs were divided into two multiplexessuch as to allow a hierarchical typing strategy. Multiplex1 covers haplogroups BCDEF, B, C, DE, D, E, F, F3, G,H, IJKLT, I, J and KLT. If a sample is found to belong tothe latter, it can subsequently be typed with multiplex 2which covers haplogroups KLT, K, K1, K2, K3, K4, M,N, O, P, Q, R, R-M412 (also known as R1b1a2a1a), Sand LT.

To maintain high sensitivity of the multiplexes, PCRamplicons were kept short with an average length of103 bp (minimum, 46 bp; maximum, 178 bp). Therecommended amount of template DNA for the PCRreactions is 12 ng, which gives satisfactory resultswhen the DNA is of reasonable quality (Fig. 2).Although we did not further evaluate the sensitivity ofthe two multiplex assays, we expect that in many cases,lower amounts of template DNA will still yield informa-tive genotypes.

Int J Legal Med

Table1

Genotypingdetails

ofY-SNPmultip

lex1

Locus

Mutation

PCRam

plification

Single-base

extension

Primer

sequences(5

-3)

Conc.

(M)

Amplicon

size

(bp)

Primer

sequence

(53)(5

aspecifictail

inlowercase

italics)

Conc.

(M)

Length

(nt)

Orientatio

nAlleles

(dye)

M91

insT

FCAAAAAT

CCCCCTA

CAT

TGC

0.600

144/143

gCTA

CAGTA

GTGAACTGAT

TAAAAAAAA

0.300

28R

a(yellow),i(green)a

RGCAGTGCCCTTCCAAATAAA

0.600

M60

insT

FTCTTTA

CAT

TTCAAAAT

GCAT

GACT

0.600

128/129

ct(gac

t)6TA

ACCACTGTGTGCCTGAT

0.600

45R

a(yellow),i(green)a

RGAGAAGGTGGGTGGTCAAGA

0.600

M145

G->A

FGCATACTTGCCTCCACGACT

0.200

96ct(gac

t)3ga

cTA

GGCTA

AGGCTGGCTCT

0.450

35R

G(yellow),A

(red)

RCCTCCCACTCCTTTTTGGAT

0.200

M174

T->C

FTCTCCGTCACAGCAAAAAT

G0.450

178

ct(gac

t)5gATACCTTCTGGAGTGCCC

0.100

41F

T(red),C(yellow)

RAGGAGAAGGACAAGACCCAT

C0.450

M96

G->C

FTGAGCTGTGAT

GTGTA

ACTTGG

0.200

117

act(ga

ct) 1

0ga

cTGGAAAACAGGTCTCTC

ATAATA

0.200

69F

G(blue),C(yellow)

RCACCCACTTTGTTGCTTTGT

0.200

M216

C->T

FCCTCAACCAGTTTTTATGAAGCTA

0.100

102

ct(gac

t)6gCTGCTA

GTTATGTATA

CCTGTT

GAAT

0.075

53R

C(blue),T(green)

RTTCTA

AAT

CTGAAT

TCTGACACTGC

0.100

M89

C->T

FCAGCTTCCTGGAT

TCAGCTC

0.200

105

ct(gac

t)13ga

AACTCAGGCAAAGTGAGAGAT

0.300

77R

C(blue),T(green)

RCACTTTGGGTCCAGGAT

CAC

0.200

M282

A->G

FTGTGCAACCTCAACTTTGCTT

0.750

106

t(ga

ct) 1

5GAAAGCAAAAT

CTCAATAT

GATAA

1.000

85F

A(green),G

(blue)

RTGTGAT

CAACTTCTTTCCCTCA

0.750

P257

G->A

FACCCCTCAGTCTCTCCGAT

T0.200

71(gac

t)9gAT

TATCCCACTGCAT

TTCTG

0.300

57F

G(blue),A

(green)

RTCAT

CTCCAACCCCCAT

CT

0.200

M69

T->C

FGGAGGCTGTTTA

CACTCCTGA

0.300

87(gac

t)10gGGCTGTTTA

CACTCCTGAAA

0.150

61F

T(red),C(yellow)

RTCTCCCCTTA

GCTCTCCTGTT

0.300

M522

G->A

FTCCAAT

TCCCAT

GTCCTCTC

0.100

109

t(ga

ct) 11CTA

CTA

CGCCTCTCTTGTCC

0.075

65F

G(blue),A

(green)

RCAGTGCAGAAAAT

CACGGTA

GA

0.100

M258

T->C

FTTCAGGAT

TTGTCAAGGAT

GG

0.200

108

t(ga

ct) 3ga

cGGGAT

TCCAAGTTCCCA

0.300

33R

T(green),C(blue)

RGCTATGACTA

AGAGGGAT

TCCAA

0.200

M304

A->C

FTTGTA

ACAAACAGTATGTGGGAT

TT

0.200

88ac

t(ga

ct) 11ga

TTATA

CCAAAATAT

CACC

AGTTGT

0.300

73R

A(red),C(blue)

RCGTCTTATA

CCAAAATAT

CACCAGTT

0.200

M9

C->G

FCTGCAAAGAAACGGCCTA

AG

0.100

90t(ga

ct) 7gCGGCCTA

AGAT

GGTTGAAT

0.100

49F

C(yellow),G

(blue)

RAACTA

AGTATGTA

AGACAT

TGAA

CGTTTG

0.100

aaancestral,iinsertion

Int J Legal Med

Table2

Genotypingdetails

ofY-SNPmultip

lex2

Locus

Mutation

PCRam

plification

Single-base

extension

Primer

sequences(5

3)

Conc.

(M)

Amplicon

size

(bp)

Primer

sequence

(53)(5

aspecifictail

inlowercase

italics)

Conc.

(M)

Length

(nt)

Orientatio

nAlleles(dye)

M9

C->G

FCTGCAAAGAAACGGCCTA

AG

0.300

90t(ga

ct) 7gCGGCCTA

AGAT

GGTTGAAT

0.100

49F

C(yellow),G

(blue)

RAACTA

AGTATGTA

AGACAT

TGAACGTTTG

0.300

M526

A->C

FTA

GAGGCAGGGTGTTGCTCT

0.300

100

ct(gac

t)10ga

TGTCAT

CAGGCTGAAT

CATAC

0.450

65F

A(green),C(yellow)

RTA

CTTTGGGAGGCTGCTGTT

0.300

M147

insT

FCCTGAATAAGCTGGTGAAAGAAA

0.500

114/115

ct(gac

t)11ga

CCTGTCTCTGAAAGAAAAAAA

1.000

69R

a(yellow),i(green)a

RGGAGACCCTGTCTCTGAAAGAA

0.500

P308

C->T

FGCTA

CCAATACCCCCAAAGA

0.050

108

gactga

cGAAAT

GAT

TAAGTA

AGTGCCTTCT

0.150

31R

C(blue),T(green)

RCCTGGAATAT

GGCACGAAAT

0.050

P79

T->C

FTTGCTTA

GTATA

ATGTCTTTCAT

GCTC

0.500

101

ct(gac

t)4gTGCTCAT

TCGCAT

CTTTG

1.000

37F

T(red),C(yellow)

RAAAT

GAGGCTA

ATCAAT

GGAACA

0.500

P261

G->A

FTCCTA

GAAGGTA

ACCCACTA

CCC

0.500

93t(ga

ct)7ga

cTTTTTGTTTTTATTA

ATGAAT

GCTA

1.000

57R

G(yellow),A

(red)

RTGTGCATAT

GTTATCCACCAT

GT

0.500

P256

G->A

FTCTTGGTTTTCCCAT

TGACC

0.200

91t(ga

ct) 1

3ga

TGCCCTA

CACTA

GATAGAAAGG

0.150

77F

G(blue),A

(green)

RCAT

CTCCCAACTTGTCTGTGC

0.200

M231

G->A

FAACAACAT

TTA

CTGTTTCTA

CTGCTTTC

0.300

119

act(ga

ct) 3gCGAT

CTTTCCCCCAAT

T0.450

33R

G(yellow),A

(red)

RTTCACAT

CAT

CCAGTA

CAGCAA

0.300

M175

5bp

del

FCCCAAAT

CAACTCAACTCCAG

0.300

101/96

t(ga

ct) 1

0CACAT

GCCTTCTCACTTCTC

0.600

61F

a(red),d(green)a

RTTCTA

CTGATACCTTTGTTTCTGTTCA

0.300

M45

G->A

FCAT

CGGGGTGTGGACTTTA

0.400

109

act(ga

ct) 6gAAT

TGGCAGTGAAAAAT

TATAGATA

0.750

53F

G(blue),A

(green)

RCCTCAGAAGGAGCTTTTTGC

0.400

M242

C->T

FAAAAAGGTGACCAAGGTGCT

0.400

46ct(gac

t)6gCGTTA

AGACCAAT

GCCAA

0.100

45R

C(blue),T(green)

RAAAAACACGTTA

AGACCAAT

GC

0.400

M207

A->G

FGGGGCAAAT

GTA

AGTCAAGC

0.300

83(gac

t)14gAAT

GTA

AGTCAAGCAAGA

AAT

TTA

0.300

81F

A(green),G

(blue)

RTCACTTCAACCTCTTGTTGGAA

0.300

M412

G->A

FGGCACTCCTCCGTCAT

CTT

0.300

114

ct(gac

t)16GGGTA

CAAT

CTGAT

GAGGC

0.300

85F

G(blue),A

(green)

RGGTGAAGTGGACCCTATCCA

0.300

P202

T->A

FAAACTTCCCAGTTTGTGGTTC

0.300

125

ct(gac

t)12ga

CCAGTTTGTGGTTCTTTGTTA

0.300

73F

T(red),A

(green)

RTGAT

CCCTTA

ATTA

ATAGCAAGACC

0.300

P326

T->C

FTTCAGATAT

CAGGCCGCTTT

0.200

61t(ga

ct) 3

CCTA

AGCAGAGGAAAATA

GTA

CAG

0.150

37R

T(green),C(blue)

RGAGCTGTCAGCCTGCCTA

AG

0.200

aaancestral,iinsertion,

ddeletio

n

Int J Legal Med

The multiplexes were optimized on a Genetic Analyzerusing POP-7 polymer. We noticed in the past that the typeof POP polymer has some influence on the relativeelectrophoretic mobilities as well as peak intensities of theextended fragments. Therefore, re-adjustment of 5 taillengths as well as reaction concentrations of the extensionprimers might be necessary when employing a POPpolymer that is different from the one used here.

Conclusion

The multiplex assays presented here form a convenient tool fordetecting the major worldwide Y haplogroups, hence giving afirst idea about the patrilineal bio-geographic ancestry of men,being of relevance in forensic investigation and anthropolog-ical research. Notably, for most of the haplogroups coveredhere, more detailed phylogenetic resolution can be obtained by

Fig. 1 Y-SNP marker phylogeny, inferred haplogroups and theirgeographic distributions as covered by the two Y-SNP multiplexassays introduced here. The phylogeny shown is a truncation of the

entire Y-chromosome tree. Due to the fact that some haplogroups haveso far been observed only sporadically, their regions of occurrence areless certain and are therefore shown in parentheses

Int J Legal Med

genotyping additional Y-SNPs. Hence, we foresee thatadditional multiplex assays, targeting more downstream Y-SNPs and dedicated to the dissection of particular (sub)haplogroups, will form useful additions to the global assayspresented here. For more complete reconstruction of a person's

overall bio-geographic ancestry, we recommend that Y-chromosome markers are combined with ancestry-informative markers from mitochondrial DNA and autosomalDNA, as already achievable with efficient multiplex toolsoffering resolution on a continental level [e.g. 7, 13].

Fig. 2 Typical electropherograms obtained with the two Y-SNPmultiplex assays introduced here, using DNA samples belonging toa range of Y haplogroups. For each peak, the detected allele is

indicated in concordance with Tables 1 and 2. As is convention, theyellow dye is shown as black for better contrast

Int J Legal Med

Acknowledgements We thank Qasim Ayub and Chris Tyler-Smithfor help in providing access to DNA samples belonging to various Yhaplogroups. This work was supported in parts by funding providedby the Netherlands Forensic Institute (NFI) and by the NetherlandsGenomics Initiative (NGI)/Netherlands Organization for ScientificResearch (NWO) within the framework of the Forensic GenomicsConsortium Netherlands (FGCN).

Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License which per-mits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.

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An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroupsAbstractIntroductionMaterials and methodsDNA samplesPrimer designPCR amplificationSingle-base extensionCapillary electrophoresis

Results and discussionConclusionReferences

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