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Text S1.
Amplification and minisequencing protocols
Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3) was used for designing the primers both for PCR amplification and minisequencing reaction. All of them have an annealing temperature around 60 C. The sequence databases at the National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) were interrogated using BLAST in order to test the primers against possible repetitive sequences and sequence homologies in the nuclear genome. Each primer pair for PCR amplification and each single base extension primer were selected independently and AutoDimer (http://www.cstl.nist.gov/biotech/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm) was used to test for potential hairpin structures and primer-dimer problems.
The SNPs were amplified in three independent PCR multiplex reactions. The size of the amplicons ranges from 66 and 195 bp. Some SNPs are located in the same amplicon, reducing substantially the number of amplicons needed in the whole multiplex design. We performed each multiplex reaction using 5 ng of DNA template and PCR master mix of QIAGEN Kit Multiplex PCR (Qiagen, Dusseldorf, Germany), amplification primers and their final concentrations are in Table 1 to Table 3 below. Amplification was carried out in a 9700 Thermocycler (Applied Biosystems, Foster City, CA, USA). After a 95 C pre-incubation step for 15 minutes, PCR was performed in a total of 30 cycles using the following conditions: 94 C denaturation for 30 seconds, annealing at 58 C for 90 seconds and extension at 72 C during 90 seconds, followed by a 15 minutes of final extension at 72 C and 4 C until removed from thermocycler.
Previous to minisequening reaction, PCR products were treated with ExoSAP-IT (Amershan Biosciences, Uppsala, Sweden) to remove excess of primers and u n - i n c o r p o r a t e d d N T P s : 1 l o f P C R p r o d u c t w a s i n c u b a t e d t o g e t h e r w i t h 0 . 5 l o f E x o S A P - I T f o r 1 5 m i n u t e s a t 3 7 C f o l l o w e d b y 1 5 m i n u t e s a t 8 5 C f o r e n z y m e i n a c t i v a t i o n . M i n i s e q u e n c i n g r e a c t i o n w a s p e r f o r m e d u s i n g S N a P s h o t T M K i t ( A B ) . M i n i s e q u e n c i n g p r i m e r s r a n g e i n s i z e f r o m 2 1 t o 8 6 b p s , l e n g t h p r i m e r s w e r e m o d i f i e d b y t h e a d d i t i o n o f n o n - h o m o l o g o u s t a i l s , p o l y ( d G A C T ) a d d e d a t t h e 5 - e n d ( T a b l e 4 t o T a b l e 6 ) . T h e m i n i s i q u e n c i n g r e a c t i o n w a s c a r r i e d o u t i n a t o t a l v o l u m e o f 1 0 l c o m p r i s i n g 3 l o f t h e S N a P s h o t T M K i t ( A B ) , 1 . 5 l P C R p r o d u c t , 1 l o f e x t e n s i o n p r i m e r s m i x ( f i n a l c o n c e n t r a t i o n s a r e b e t w e e n 0 . 0 6 a n d 0 . 6 M ) , a n d w a t e r - u p t o 1 0 l . T h e r e a c t i o n w a s p e r f o r m e d i n a 9 7 0 0 T h e r m o c y c l e r ( A B ) f o l l o w i n g t h e r e c o m m e n d a t i o n s o f t h e m a n u f a c t u r e r : 2 5 c y c l e s o f d e n a t u r a t i o n a t 9 6 C f o r 1 0 s e c o n d s , a n n e a l i n g a t 5 0 C f o r 5 s e c o n d s a n d e x t e n s i o n a t 6 0 C d u r i n g 3 0 s e c o n d s . U n - i n c o r p o r a t e d d d N T P s w e r e e l i m i n a t e d u s i n g S A P ( A m e r s h a n B i o s c i e n c e s ) . T h e f i n a l v o l u m e ( 1 0 l ) w a s t r e a t e d w i t h 1 l o f S A P f o r 6 0 m i n u t e s a t 3 7 C f o l l o w e d b y 1 5 m i n u t e s a t 8 5 C f o r e n z y m e i n a c t i v a t i o n .
T h e m i n i s i q u e n c i n g p r o d u c t s ( 2 l ) w e r e m i x e d w i t h 9 . 5 l o f H i D i T M f o r m a m i d e ( A B ) a n d 0 . 3 l o f G e n e S c a n 1 2 0 L I Z ( A B ) a n d c a p i l l a r y e l e c t r o p h o r e s i s w a s u n d e r t a k e n o n a n A B I P R I S M 3130xl Genetic Analyzer (AB) .Resulting data were analyzed using GeneMapperTM 3.7 Software (AB).
Figure 1 below shows an example of a minisequencing electropherogram.
Automatic sequencing of control region
All the samples from Galicia, Cantabria, and Catalonia (N=518) were sequenced for the HVS-I. PCR amplification was carried out in a 9700 Thermocycler (AB). The temperature profile for 32 cycles of amplification was 95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 30 seconds. Sequencing primers were previously described by ADDIN EN.CITE Wilson19952536253617Wilson, M. R.DiZinno, J. A.Polanskey, D.Replogle, J.Budowle, B.Forensic Science Research and Training Center, FBI Academy, Quantico, VA 22135, USA.Validation of mitochondrial DNA sequencing for forensic casework analysisInt J Legal MedInt J Legal Med68-741082AnimalsBase SequenceBlood StainsDNA Fingerprinting/*standardsDNA, Mitochondrial/*geneticsHairHumansImmunoglobulin Variable RegionMolecular Sequence Data*Polymerase Chain ReactionReproducibility of ResultsSemenSequence Analysis, DNA/*methodsSpecimen Handling19958547161http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8547161 (Wilson et al. 1995). PCR product purification and sequencing were performed as in Salas et al. ADDIN EN.CITE Salas19981007100717Salas, A.Comas, D.Lareu, M. V.Bertranpetit, J.Carracedo, .Institute of Legal Medicine, University of Santiago de Compostela, Spain.mtDNA analysis of the Galician population: a genetic edge of European variationEur J Hum GenetEur J Hum Genet365-7564Base SequenceDNA PrimersDNA, Mitochondrial/*geneticsHumanMolecular Sequence DataSequence Homology, Nucleic AcidSpainSupport, Non-U.S. Gov't*Variation (Genetics)1998Jul-Aug9781045http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9781045(1998). For those sequences containing a continuous homopolymeric cytosine stretch from positions 16184 to 16193 (usually related to length heteroplasmy), additional amplification and sequencing were performed in order to fully cover this region.
Genotyping transition C4592T
Using the same protocol described above (for primers see the corresponding amplicon 4550-4580 described in Table 1), transition C4592T was sequenced in all the haplogroup H2 mtDNAs observed in our samples from Galicia, Cantabria, and Catalonia. None of them carried this transition.
Complete genome genotyping
The eight samples collected in the Basque Country belonging to H2a5 were sequenced for the complete genome. Primers used for PCR amplification and sequencing were those reported in ADDIN EN.CITE Torroni20011060106017Torroni, A.Rengo, C.Guida, V.Cruciani, F.Sellitto, D.Coppa, A.Calderon, F. L.Simionati, B.Valle, G.Richards, M.Macaulay, V.Scozzari, R.Dipartimento di Genetica e Microbiologia, Universita di Pavia, Pavia, Italy. torroni@ipvgen.unipv.itDo the four clades of the mtDNA haplogroup L2 evolve at different rates?Am J Hum GenetAm J Hum Genet1348-56696Base SequenceDNA, Mitochondrial/*geneticsDominican Republic*Evolution, MolecularHaplotypes/*geneticsHumanKineticsMutation/genetics*PhylogenyPolymerase Chain ReactionPolymorphism, Restriction Fragment LengthSupport, Non-U.S. Gov'tVariation (Genetics)/genetics2001Dec11595973http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11595973(Torroni et al. 2001). PCR was performed in 10l of reaction mix, containing 4 l of PCR Master Mix (Qiagen), 0.5 l 1M of each primer, 1 l sample template and 4 l of water. This PCR was carried out in a 9700 Thermocycler (AB) with one cycle of 95 C for 15 minutes and then 35 cycles of 94 C for 30 seconds, 58 C for 90 seconds and 72 C for 90 seconds with a full extension cycle of 72 C for 10 minutes. The PCR product was checked in agarose gels and purified using QIAquickR Gel Extraction Kit (Qiagen).
Sequencing reaction was performed in 11.5 l of reaction mixture, containing 2.5l of sequencing buffer (5X), 0.5 l of BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), 1 l of the corresponding primer (final concentration was 1 M), 3 l of the purified PCR product and water up to 11.5 l. Sequencing reaction was carried out in a 9700 Thermocycler (AB) with one cycle of 96 C for 3 minutes and then 25 cycles of 96 C for 30 seconds, 50 C for 15 seconds and 60 C for 4 minutes or was carried out in a 9800 Fast Termal Cycler (Applied Biosystems) with one cycle of 96 C for 1 minute then 25 cycles of 96 C for 10 seconds, 50 C for 5 seconds and 60 C for 1 minute. To obtain clean electropherograms, the sequencing product was doubled purified, first using MontageTM SEQ96 Sequencing Reaction Cleanup Kit (Millipore) according to manufacturer protocols, followed by purification with SephadexTM G-10 (Amershan Biosciences, Uppsala, Sweden), the latter also according to manufacturer protocol. MtDNA automatic sequencing was carried out in a capillary electrophoresis ABI3730 (Applied Biosystems).
References
ADDIN EN.REFLIST Salas A, Comas D, Lareu MV, Bertranpetit J, Carracedo (1998) mtDNA analysis of the Galician population: a genetic edge of European variation. Eur J Hum Genet 6(4): 365-375.
Torroni A, Rengo C, Guida V, Cruciani F, Sellitto D et al. (2001) Do the four clades of the mtDNA haplogroup L2 evolve at different rates? Am J Hum Genet 69(6): 1348-1356.
Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B (1995) Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 108(2): 68-74.
Table 1. Amplification primers for multiplex 1. In the SNP column indicates the SNP genotyped in each amplicon. F.C. = final concentration
SNPPrimer forward and reverseSize (bp)F.C. (M)709/750GGCTCACATCACCCCATAAA
CGTTTTGAGCTGCATTGCT1610.22581/2706GCCTGCCCAGTGACACAT
GCTCCATAGGGTCTTCTCGT1950.23010CAATAACTTGACCAACGGAACA
CGGTCTGAACTCAGATCACGTA1790.43796/3847TCAACATTACTAATAAGTGGCTCCTTT
GGTTCGGTTGGTCTCTGCTA1350.24550/4580CAACCCGTCATCTACTCTACCAT
CTTCTGTGGAACGAGGGTTTATT1480.26253/6296/6365TGACTCTTACCTCCCTCTCTCC
GATGAAATTGATGGCCCCTA1890.26776GCTTCCTAGGGTTTATCGTGTG
GAGTGTGGCGAGTCAGCTAAA1400.47337GGCTCATTCATTTCTCTAACAGC
TCCAGGTTTATGGAGGGTTC1100.410810GCTAAAACTAATCGTCCCAACA
AATTAGGCTGTGGGTGGTTG970.212858/12957CAACACAGCAGCCATTCAAG
GAGGCCTAGTAGTGGGGTGA1570.213708/13759 AACGAAAATAACCCCACCCTA
GTTGTTTGGAAGGGGGATG1130.214365/14470CCACCCCATCATACTCTTTCA
TAGGGGGAATGATGGTTGTC1590.314766/14770ATCAACTACAAGAACACCAATGACC
GGAGGTCGATGATGAGTGG820.215218ACTATCCGCCATCCCATACA
GGGCAAGATGAAGTGAAAGG1100.4
Table 2. Amplification primers for multiplex 2. In the SNP column indicates the SNP genotyped in each amplicon. F.C. = final concentration
SNPPrimer Forward and ReverseSize (bp)F.C. ( M ) 9 5 1 / 9 6 1 G T C A C A C G A T T A A C C C A A G T C A
A C T C A G G T G A G T T T T A G C T T T A T T G 8 7 0 . 2 3 9 1 5 / 3 9 3 6 / 3 9 9 2 T A G C A G A G A C C A A C C G A A C C
G A A G A T T G T A G T G G T G A G G G T G T 1 5 8 0 . 6 4 3 1 0 / 4 3 3 6 A G C A T T C C C C C T C A A A C C T A
T T T T G G A T T C T C A G G G A T G G 1 2 7 0 . 4 4 7 2 7 / 4 7 4 5 / 4 7 6 9 / 4 7 9 3 T C C T T C T A A T A G C T A T C C T C T T C A A C A
T G G G T A A C CTCTGGGACTCA1540.47028CACCGTAGGTGGCCTGACTGGC
GTGTAGCCTGAGAATAGGGG1680.47645ACATGCAGCGCAAGTAGGTC
AAAATGATTATGAGGGCGTGA900.28269/8271TTAGGGCCCGTATTTACCCTAT
AAGAGGTGTTGGTTCTCTTAATCTTT1100.28473/8592/8598/8602CCCAACTAAAAATATTAAACACAAACT
GGAGGTGGGGATCAATAGAG1930.29066/9088/9150CCTACTCATGCACCTAATTGGA
GGCTTACTAGAAGTGTGAAAACGTA1550.310044CCGTTAACTTCCAATTAACTAGTTTTG
AAGGCTAGGAGGGTGTTGATT910.610394CCATGAGCCCTACAAACAACT
TGAGTCGAAATCATTCGTTTTG1590.313404TATGTGCTCCGGGTCCATC
TGGTGAG G G A G G T T G A A G T G 1 0 4 0 . 2
T a b l e 3 . A m p l i f i c a t i o n p r i m e r s f o r m u l t i p l e x 3 1 . I n t h e S N P c o l u m n i n d i c a t e s t h e S N P g e n o t y p e d i n e a c h a m p l i c o n . F . C . = f i n a l c o n c e n t r a t i o n
S N P P r i m e r F o r w a r d a n d R e v e r s e S i z e ( b p ) F . C . ( M ) 1 4 3 8 A A C T T A A G G G T C G A A GGTGGA
AGGGCCCTGTTCAACTAAGC660.12259TCAAGCTCAACACCCACTACC
TGCGGAGGAGAATGTTTTCA1310.25250/5263ATTCCATCCACCCTCCTCTC
GGTGGGGATGATGAGGCTAT1110.68869GGACTCCTGCCTCACTCATTT
AAGTGGGCTAGGGCATTTTT1280.48994AATGCCCTAGCCCACTTCTT
AGGTGGCCTGCAGTAATGTT1400.19336GCCATGTGATTTCACTTCCA
GTGGCCTTGGTATGTGCTTT1170.210166/10211ACTACCACAACTCAACGGCTACA
AGGGGTAAAAGGAGGGCAAT1450.211140CATTCACAGCCACAGAACTAATCAT
GTTCTGGCTGGTTGCCTCAT990.211719CAGCCATTCTCATCCAAACC
GCGTTCGTAGTTTGAGTTTGC1130.312308CTGCTAACTCATGCCCCCATG
ATTACTTTTATTTGGAGTTGCACCAAGATT1060.312438CCACCCTAACCCTGACTTCC
GTGGATGCGACAATGGATTT1060.112705TGTAGCATTGTTCGTTACATGG
AGTTGGAATAGGTTGTTAGCGG1470.213101C/13105CAGCCCTACTCCACTCAAGC
TGGGCTATTTTCTGCTAGGG830.214869/14872CAACATCTCCGCATGATGAA
AGGCGTCTGGTGAGTAGTGC1040.215452AAGACGCCCTCGGCTTACTT
GTCGCCTAGGAGGTCTGGTG780.215773CCGCAGACCTCCTCATTCTA
CGGATGCTACTTGTCCAATG810.115833/15904CCCTTTTACCATCATTGGACA
AAAGGTTTTCATCTCCGGTTT1620.2
Table 4. Minisequencing primers for multiplex 1. In the SNP column indicates the SNP genotyped in each amplicon. F.C. = final concentration
SNPMinisequencing primerSize (pb)Base changeChainF.C. (M)709(gact)3TTACACATGCAAGCATCCCC32G-AL0.2750CTCTAAATCACCACGATCAAAAGG24A-GL0.22581TGATTATGCTACCTTTGCACGGT23A-GH0.22706(gact)2gAGGGTCTTCTCGTCTTGCTGTGT32A-GH0.23010 (gact)3gAACCTTTAATAGCGGCTGCACCAT37G-AH0.23796(gact)2gaCTAATAAGTGGCTCCTTTAACCTCTCC37A-GL0.13847(gact)5ATTACTCCTGCCATCATGACCC42T-CL0.24550(gact)13gaGCGCTAAGCTCGCACTGATT74T-CL0.34580(gact)3gaTTACCTGAGTAGGCCTAGAAATAAACAT42G-AL0.26253(gact)6gaTGTTCCTGCTCCGGCCTCCACT48T-CH0.16296(gact)6AACAGGTTGAACAGTCTACCCTCC48C-TL0.26365(gact)7gaGATGGCCCCTAAGATAGAGGAGAC54T-CH0.26776(gact)6acCGTGTGTCTACGTCTATTCCTACTGTAAATAT58T-CH0.37337(gact)10TGATTTGAGAAGCCTTCGCTTC62G-AL0.310810(gact)5gacCAACAATTATATTACTACCATTGACATGACT54T-CL0.212858(gact)8gacGCAGCCATTCAAGCAATCCTATA58C-TL0.312957(gact)9CAACAAATAGCCCTTCTAAACGCTAA62T-CL0.313708(gact)13 gaCTACTAAACCCCATTAAAGGCCTG78G-AL0.313759(gact)10TTCTCATTACTAACAACATTTCCCCC66G-AL0.114365(gact)11 gaGTTAGCGATGGAGGTAGGATTGGT70C-TH0.314470A(gact)13gCCTCAATAGCCATCGCTGTAGTATA78T-AL0.414770(gact)11GAATGAGTGGTTAATTAATTTTATTAGGGG74C-TH0.314766(gact)11 gCAATGACCCCAATACGCAAAA66T-CL0.315218(gact)11TCCTCAGATTCATTGAACTAGGTCTG70A-GH0.3
Table 5. Minisequencing primers for multiplex 2. In the SNP column indicates the SNP genotyped in each amplicon. F.C. = final concentration
SNPMinisequencing primerSize (pb)Base changeChainF.C. (M)951CTTTATTGGGGAGGGGGTGAT21G-AH0.2961G(gact)15CGTAAAGAGTGTTTTAGATCACCCCC86T-GL0.063915(gact)2GAAGCCTGAGACTAGTTCGGACTC32G-AH0.23936(gact)14gTGCGGCGTATTCGATGTTGAA78C-TH0.63992(gact)3gaCCCTATTCTTCATAGCCGAATACA38C-TL0.34310TCTGATAAAAGAGTTACTTTGATAGAGTAAATAATAGG38A-GL0.44336AGGGATGGGTTCGATTCTCAT21T-CH0.24727(gact)5gaTACTCTCCGGACAATGAACCAT44A-GL0.24745gactAATGAACCATAACCAATACTACCAATCA32A-GL0.24769(gact)4gaACCAATCAATACTCATCATTAATAATCATAAT50A-GL0.24793(gact)5gATAATCATAATAGCTATAGCAATAAAACTAGGAAT56A-GL0.47028(gact)4gacTACACGACACGTACTACGTTGTAGC44C-TL0.27645(gact)6GCTACTTCCCCTATCATAGGAGAGCT50T-CL0.28269(gact)8gacTGAAATAGGGCCCGTATTTACCCTATA62G-AL0.068271T(gact)15GGGCCCGTATTTACCCTATAGC82A-TL0.38473(gact)6gaAAAAATATTAAACACAAACTACCACCTACC56T-CL0.28592(gact)11gacGCCTACCCGCCGCAGTACT66G-AL0.18598(gact)15gacACCCGCCGCAGTACTGATCAT84T-CL0.48602(gact)13 CGCCGCAGTACTGATCATTCTA74T-CL0.49066(gact)8gaGTGTAGAGGGAAGGTTAATGGTTGATAT62A-GH0.39088(gact)10AGTAGAATTAGAATTGTGAAGATGATAAGTGTAG74T-CH0.29150(gact)12gCCTAGAAATCGCTGTCGCCTT70A-GL0.210044(gact)9TTAAGGCGAAGTTTATTACTCTTTTTTGAA 66A-GH0.610394(gact)9gaCTGGCCTATGAGTGACTACAAAAAGGATTAGA70C-TL0.313404(gact)11gaATCATCCACAACCTTAACAATGAACAAGATAT78T-CL0.3
Table 6. Minisequencing primers for multiplex 3. In the SNP column indicates the SNP genotyped in each amplicon. F.C. = final concentration
SNPMinisequencing primerSize (pb)Base changeChainF.C. (M)1438(gact)10gaGTCGAAGGTGGATTTAGCAGTAAACT68A-GL0.062259TCCCAAACATATAACTGAACTCCTCA26C-TL0.25250TTCTTCGATAATGGCCCATTTGGGCA 26T-CH0.25263(gact)2gacGGCTTTTTGCCCAAATGGG30C-TL0.28869(gact)2gTTATGAGCGGGCACAGTGATT30A-GL0.68994(gact)9gacGCCTACTCATTCAACCAATAGCCCT64G-AL0.069336(gact)3gacTTCCACTCCAT 4 F m n
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