DNA concentration

The quality of the extracted samples was then assessed using the eight-channel NanoDrop 8000 (NanoDrop Technology^®, Cambridge, UK), a UV micro-spectrophotometer sample retention system that calculates the concentration of DNA present in the sample. First, the sample was left to reach room temperature before being vortexed to ensure a homogeneous sampling. Next, both the top and bottom portions of the fibre optic pedestal were scrubbed vigorously with a nonabrasive, anti-static, low lint cellulose fibre wipe to remove any residue from the previous use. After opening the accompanying NanoDrop 8000 software on a connected laptop, 1 μl of deionised water was placed on the lower portion of the pedestal. The lever arm was then lowered, resting close enough to create a column of liquid between both lower and upper fibre optic receptors. After initialising the machine, the receptors were then scrubbed before adding 1 μl Buffer AE to the pedestal to blank the system by lowering the lever arm and initialising the machine again. The surface was scrubbed once more and the system made ready to measure the quality of the samples. Extracted DNA (1 μl) was pipetted onto the pedestal, the lever arm lowered and the machine initialised. After the values were recorded using the software, the fibre optic pedestals were scrubbed clean in preparation for the next batch. Each sample was

repeated at least twice to give a more accurate measure of the amount of DNA present in the sample.

The above qualification was especially important to the whole-genome sequencing mentioned in Section 5.2.1, where the amount of DNA present in each sample was critical to generate high quality output. Rather discouragingly, the values generated using the NanoDrop 8000 produced a weak relationship with the values later quantified by the PicoGreen method (Invitrogen Ltd., Paisley, UK) used by collaborators at the Tokyo Metropolitan Institute of Gerontology. This is plotted in Figure 2.1. The main disadvantage of the Nanodrop UV spectroscopy system used above was the inclusion of single-stranded DNA and other contaminants such as proteins and extraction buffers in the estimate of double-stranded DNA concentration (Keer & Birch, 2008). The PicoGreen reagent binds to double-stranded DNA and creates a maximum emission at 530 nm and has been shown to provide not only better measurement accuracy but also better consistency (English et al., 2006). The observed pattern of dispersal may also indicate that the DNA was not fully dissolved into the buffering solution.

Figure 2.1 Comparison of DNA concentrations generated by the NanoDrop microspectrophotometer and PicoGreen assay. Each point represents an individual sample. The coefficient of determination (𝑟²) indicates that the values generated for the NanoDrop were only able to explain 46.7% of the variance. Shaded area represents a 95% confidence interval for the line of best fit, shown in blue.

2.3 Mitochondrial DNA

HVS-1 was amplified and sequenced to identify patterns of polymorphisms, known as HVS-1 motifs. The reverse strands of mtDNA were sequenced using fluorescent dideoxynucleotides (ddNTPs) in the BigDye™ terminator cycle sequencing ready reaction (Applied Biosystems, Foster City, CA, USA). A reverse primer was used to generate all sequences and when necessary, a forward primer was also used to read the sequence on the 5’ end of the polycytosine tract between base pairs 16184-16193, according to the revised Cambridge reference sequence for mtDNA (rCRS (Andrews et al., 1999), NCBI Reference Sequence: NC_012920.1). These primers can be found in Table 2.2. HVS-1 polymerase chain reaction (PCR) sequencing was performed from a 20 µl total volume, containing 10µL of 2x ReddyMix PCR master mix (Thermo Scientific, Surrey, UK), 1 µl of 0.2 µM primer and 2 µl of total DNA (estimated at 10 ng, based on Qiagen QIAamp DNA Mini kit documentation and tested averages). The remaining 6 µl were composed of autoclaved, deionised, ultraviolet-treated water. DNA was denatured for 5 minutes at 94ºC followed by 30 cycles of denaturation (30 seconds, 94ºC), annealing (45 seconds, 51ºC) and elongation (60 seconds, 72ºC). A final elongation step was performed (10 minutes, 72ºC) before samples were held at 4ºC. Negative controls with no DNA were included in each sample run to aid in the detection of contamination. PCR product was then run on a 2% agarose electrophoresis gel containing ultraviolet-fluorescent nucleic acid stain ethidium bromide to determine whether the reaction had been successful. Sequencing products were separated by 5% denatured Long Ranger™ gel (FMC Bio-Products, Rockland, ME, USA) and base pairs were detected using a PE Applied Biosystems 377 DNA sequencer. Sequencing was performed by the Julie Galbraith of the Sir Henry Wellcome Functional Genomics Facility at the University of Glasgow.

The resulting sequence electropherograms were read using Chromas (version 1.45, Technelysium, Australia). The length polymorphism found between base pairs 16184-16193 was controlled for by manually inserting or removing cytosines to standardise the overall sequence length. Differences from rCRS were recorded manually to establish the HVS-1 motif necessary to haplogroup each sample.

For further resolution of ambiguous haplotype motifs, RFLPs were used to genotype coding region mtSNPs. Three mtSNPs were chosen to refine African haplogroups based on the phylogenetic tree of recorded global whole-genome

mitochondrial diversity (van Oven & Kayser, 2009). The transition G10398A was used here predominately to resolve macro-haplogroup N, though it can be used to distinguish finer-resolution of Sub-Saharan haplogroups L0d1b1, L1c1a1 and L3e1a3. The transition C10400T was used to resolve macro-haplogroup M. Finally, the transition 13803 was used to resolve the Sub-Saharan haplogroup L2. Primers were designed using the NCBI Primer-BLAST (Ye et al., 2012) and checked against the revised Cambridge Reference Sequence of human mtDNA (rCRS) for any typographic errors (Table 2.2). PCR cycles were identical to that of the HVS-1 protocol. Although the various primers’ melting temperatures were held in consideration, the annealing temperature produced satisfactory results at 51°C.

The resulting PCR products then had restriction enzymes appropriate to the mtSNP added before being left to incubate at 37°C overnight. After digestion, the product was then run on a previously described agarose electrophoresis gel to determine whether the enzyme had cleaved the PCR product, indicative of an mtSNP. This information was recorded and collated with the HVS-1 data to inform haplogroup assignment further.

The HVS-1 motif and any additional genotyping data were used to haplogroup these data in accordance with the comprehensive full mtDNA genome phylogenetic tree Phylotree Build 11 (7 Feb 2011) (van Oven & Kayser 2009) and the comprehensive mtDNA database published alongside the Genographic Project (Behar et al., 2007) at a comparable resolution to that in the literature (Salas et al., 2005b). To monitor errors that could arise from the sequencing procedure, motif inconsistencies were investigated by comparing potentially incorrect loci against a list of known problems and inconsistencies found in published sequence data (Bandelt et al., 2002; Bandelt et al., 2004a; Bandelt et al., 2004b; Salas et al., 2005a; Salas et al., 2007; Yao et al., 2009). Sequencing electropherograms were rechecked when such phylogenetic inconsistencies were observed. DNA samples were extracted again or resequenced when doubt persisted regarding the variant in question. Additional coding-region mtSNP genotyping performed as part of larger SNP array data (outlined in Section 5.2.1) also allowed for increased confidence during haplogroup assignment.