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Materials and methods

3. Materials and methods 1. Samples

3.3. Molecular analyses

3.3.6. Y-chromosome SNPs

Thirty-eight SNPs were typed to define the major male lineages. Thirty-one of them were genotyped using SNaPshot™ kit (Applied Biosystems) technique in five multiplexes.

Multiplex 1: M13, SRY1532.1, M213, M9, Tat, 92R7, M173, SRY1532.2, P25 and M70;

multiplex 2: M201, M170, M26, 12f2a, M62 and M172 (Figure 18); and multiplex 3: M78, M81 and M123, were described by Brion et al. (2005) and modified by Gomes et al. (2010).

Besides, multiplex R: L23, U106, M153, M167, U152 and M529, described in Marques et al. (2016) and multiplex Q: M242, P36.2, M346 M3, M19, M194 and M199, described by Roewer et al. (2013) were typed in the samples belonging to the R and Q haplogroups.

SNPs M1 and M269 were genotyped with conventional PCR followed by agarose gel electrophoresis; while S116, M17 and M18 were genotyped by Sanger sequencing (explained extensively in section 3.7), with the primers in Table 4. DYS458.2 was used to determine the J1 chromosomes (Myres et al., 2007).

Figure 17. Y-chromosome markers included in the Y-filer kit (Applied Biosystems, Foster City, CA, USA).


Table 4. Primers used to amplify SNPs M1, M269, S116, M17, and M18.

SNP Forward primers Reverse primers





Amplification of the multiplexed SNPs was performed in multiplex PCRs using 5 μl Qiagen Multiplex PCR kit (Qiagen), 1 μl primers, and 0.3–5 ng of genomic DNA in a final reaction volume of 10 μl.

PCR conditions for the amplification of the multiplexes were: initial incubation at 95 °C for 15 min; followed by 35 cycles at 94 °C for 30 s, 60 °C for 90 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min. Then agarose gel electrophoresis was carried out to ensure the amplification was performed correctly.

Purification of the samples was performed by mixing 1 μl PCR product and 0.80 μl ExoSAP-IT reagent. Then two incubations at 35 °C for 15 min followed by 85 °C for 15 min.

Afterwards, SNaPshot reaction was performed by adding 1 μl SNaPshot mix provided with the kit, and 1 μl SBE (single-base extension) primers in a final reaction volume of 5 μl to the purification tubes. Conditions for the SNaPshot reaction were: 25 cycles at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 30 s. Then, a final purification was conducted by adding 1 μl SAP to each tube and incubating at 37 °C for 60 min and at 85 °C for 15 min.

The SNaPshot chemistry is based on the dideoxy single-base extension of an unlabelled oligonucleotide primer (or primers). Each primer binds to a complementary template in the presence of fluorescently labelled ddNTPs and AmpliTaq® DNA polymerase. The polymerase extends the primer by one nucleotide, adding a single ddNTP to its 3´ end (Figure 19).

SNaPshot products were separated by capillary electrophoresis.


Figure 18. Multiplex 2 results showing an ancestral sample, a J2 M-172 sample, and a G-M201 sample.

Figure 19. SNaPshot technique amplification, based on SBE primers minisequencing (ABI PRISM® SNaPshot™ Multiplex kit user manual).

M170 - M172


M201 M26

-M172 +

M201 +

Multiplex 2 ancestral

Multiplex 2 J2-M172

Multiplex 2 G-M201

47 3.3.7. Mitochondrial DNA sequencing

Sequences of mitochondrial DNA were obtained by conventional PCR followed by Sanger sequencing technique. For all samples, the amplification of the D-loop was performed, for 17 selected samples, all the mitochondrial genome was sequenced.

Mitochondrial DNA amplification was performed using 5 μl Qiagen Multiplex PCR kit (Qiagen), 0.5 μl forward and reverse primers, and 20–25 ng of genomic DNA in a final reaction volume of 10 μl. A negative control was included in each PCR run.

PCR conditions for the amplification were: initial incubation at 95 °C for 15 min; followed by 35 cycles at 94 °C for 30 s, 60 °C for 90 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min. Then agarose gel electrophoresis was carried out to ensure the amplification was performed correctly.

Samples amplified correctly were purified with the MBS® Spin PCRapace (Invitek) following the manufacturer’s procedure. After quantification of purified samples, the sequencing reaction was performed for the two DNA strands (forward and reverse) separately using 0.6 μl BigDye® Terminator v.3.1 cycle sequencing premix (Applied Biosystems), 1.2 μl buffer, 1 μl primer, and 1 μl purified sample in a final reaction volume of 10 μl.

PCR conditions for the sequencing reaction were: initial incubation at 96 °C for 2 min;

followed by 35 cycles at 96 °C for 15 s, 50 °C for 9 s, and 60 °C for 120 s; and a final extension at 60 °C for 10 min.

In order to purify the samples, conventional precipitation with ethanol was performed. First, samples were stored at -20 °C for 15 min with absolute ethanol (100%) and sodium acetate 3M. Then samples were centrifuged for 30 min at 15777 g to precipitate. After removing the supernatant, pellets were washed with 70% ethanol, followed by another centrifuging step of 10 min. Samples were dried in a vacuum pump and, finally, diluted in mili-Q water for capillary electrophoresis in the genetic analyser.

Once the haplotype had been obtained for each sample, haplogroups were classified following the updated mtDNA phylogeny, PhyloTree, mtDNA tree Build 17 (http://www.phylotree.org/) using HaploGrep2 tool (van Oven, 2015; Weissensteiner et al.

2016), and assigned haplotypes were validated by EMPOP (EMP00672) (Parson and Dür, 2007; http://empop.org/) curators.

48 D-loop

The entire D-loop (non-codifying control region) was amplified (position 16024 to 576).

Amplifications were conducted in two fragments called F1 and F2 whose primers are specified in Table 5. Amplification and sequencing was carried out using the same primers.

Table 5. Primers used to amplify the D-loop region of mtDNA.

Forward primers Reverse primers

D-loopF1 L15997 CACCATTAGCACCCAAAGCT H016 CCCGTGAGTGGTTAATAGGGT (Alonso et al., 2003) (Parson and Bandelt, 2007)

D-loopF2 L16555 CCCACACGTTCCCCTTAAAT H639 GGGTGATGTGAGCCCGTCTA (Marques et al., 2015) (Parson and Bandelt, 2007) Complete mtDNA genome

Attempting to go further into the classification of female lineages, complete mitogenomes were sequenced in those cases that could be relevant for the study. Primers described by Ramos et al. (2009) were used to redesign a strategy of amplification in order to attain shorter, easily amplifiable, fragments. Nineteen overlapping fragments were amplified (Table 6) and finally 31 inner fragments were sequenced by Sanger technique to obtain the whole genome.

Table 6. Primer pairs from Ramos et al. (2009; 2011) used to amplify mtDNA complete genomes.

Fragment Primer pair Fragment Primer pair

MTT1A 14898for/15826rev MTT5B 7713for/9220rev

MTT1B 15416for/151rev MTT6A 8910for/10154rev

MTT2A 16488for/1159rev MTT6B 9874for/10648rev

MTT2B 909for/1677rev MTT7A 10360for/11673rev

MTT3A 1404for/2801rev MTT7B 11461for/12226rev

MTT3B 2646for/3947rev MTT8A 11977for/13297rev

MTT4A 3734for/5017rev MTT8B 12988for/13830rev

MTT4B 4896for/6154rev MTT9A 13477for/14838rev

MTT4C 5995for/6739rev MTT9B 14440for/15349rev

MTT5A 6511for/8000rev

49 3.3.8. Mitochondrial DNA SNPs

SNPs were genotyped by sequencing in those cases that could be relevant to better classify female lineages.

In the samples belonging to haplogroups R0a, K1a1b1a, T1a, and T2c1d, key mutations found in the mitogenomes amplified (Table 7) were genotyped, by sequencing the fragment containing the position of interest


Table 7. Key mutations studied in the samples belonging to haplogroups R0a, K1a1b1a, T1a, and T2c1d.

Haplogroup Key mutations

R0a 4767G, 13858G and 15734A

K1a1b1a 8029T

T1a 6656T and 10116G

T2c1d 11914A, 12363T, 1306T, 14544A, 3027C 8475T, 8911C, 8980A and 15569T

In the case of the H haplogroup samples, a set of 51 SNPs arranged in 3 multiplexes designed by Alvarez-Iglesias et al. (2009) were typed using SNaPshot technique. SNPs typed were: 709, 750, 2581, 3010, 3796, 6253, 6296, 6365, 6776, 7337, 10810, 12858, 12957, 13708, 13759, 14365, and 14470A for multiplex 1; 951, 3915, 3936, 3992, 4310, 4336, 4727, 4745, 4769, 4793, 7028, 7645, 8569, 8473, 8592, 8598, 8602, 9066, 9150, 10044, 10394, 13404, 8271T, and 961G for multiplex 2; and 1438, 2259, 8994, 10166, 10211, 11140, 14869, 14872, 15833, and 13101C for multiplex 3.