;cgacg attggggtat 530 540
ccccgaacca accaaacccc aaagacaccc ccca3agttt atgtagctta cctcctcaaa ggggcttggt tggtttgggg tttctgtggg gggtptcaaa tacatcgaat ggaggagttt 550 560 570 580 590 600
gcaatacact gaaaätgttt agacgggctc acatcacccc ataaacaaat aggtttggtc cgttatgtga cttttacaaa tctgcccgag tgtagtgggg tatttgttta tccaaaccag 610 620 630 640 650 660
Figure 10.7 Comparison of a sample with (a) 16189T (no HV1 C-stretch) to (b) one with the C-stretch. Notice how the sequence quality quickly drops after the string of cytosine residues due to the presence of two or more length variants that creates a situation where the extension products are out of phase or register with one another. Different primer combinations are typically used on samples containing a C-stretch as illustrated in (c) to recover sequence information from both strands or to provide a double read of the same strand.
Good quality sequence
Poor quality sequence
(two length variants out of phase)
(c) Primer strategies typically used with C-stretch containing samples
Use of internal primers
In the event that the C-stretch is present in a sample, different sequencing primers may be used to obtain reliable mtDNA sequence information downstream of the homopolymeric stretches (Rasmussen et al. 2002). For example, the FBI A4/B4 primer set (L16209 and H16164) shown in Figure 10.6 can be used on individuals possessing the HV1 C-stretch in order to recover sequence information from both sides of the homopolymeric stretch of cytosines (Wilson 1997). Alternatively the same strand may be examined twice in separate sequencing reactions to provide double coverage of all nucleotides (Figure 10.7c).
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