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Human Y chromosome base-substitution mutation rate measured by direct sequencing in a deep-rooting pedigree.

Xue Y, Wang Q, Long Q, Ng BL, Swerdlow H, Burton J, Skuce C, Taylor R, Abdellah Z, Zhao Y, AsanMacArthur DG, Quail MA, Carter NP, Yang H, Tyler-Smith C - Curr. Biol. (2009)

Bottom Line: Candidate mutations were further examined by capillary sequencing in cell-line and blood DNA from the donors and additional family members.Twelve mutations were confirmed in approximately 10.15 Mb; eight of these had occurred in vitro and four in vivo.The latter could be placed in different positions on the pedigree and led to a mutation-rate measurement of 3.0 x 10(-8) mutations/nucleotide/generation (95% CI: 8.9 x 10(-9)-7.0 x 10(-8)), consistent with estimates of 2.3 x 10(-8)-6.3 x 10(-8) mutations/nucleotide/generation for the same Y-chromosomal region from published human-chimpanzee comparisons depending on the generation and split times assumed.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Sanger Institute, Hinxton, Cambs CB10 1SA, UK. ylx@sanger.ac.uk

ABSTRACT
Understanding the key process of human mutation is important for many aspects of medical genetics and human evolution. In the past, estimates of mutation rates have generally been inferred from phenotypic observations or comparisons of homologous sequences among closely related species. Here, we apply new sequencing technology to measure directly one mutation rate, that of base substitutions on the human Y chromosome. The Y chromosomes of two individuals separated by 13 generations were flow sorted and sequenced by Illumina (Solexa) paired-end sequencing to an average depth of 11x or 20x, respectively. Candidate mutations were further examined by capillary sequencing in cell-line and blood DNA from the donors and additional family members. Twelve mutations were confirmed in approximately 10.15 Mb; eight of these had occurred in vitro and four in vivo. The latter could be placed in different positions on the pedigree and led to a mutation-rate measurement of 3.0 x 10(-8) mutations/nucleotide/generation (95% CI: 8.9 x 10(-9)-7.0 x 10(-8)), consistent with estimates of 2.3 x 10(-8)-6.3 x 10(-8) mutations/nucleotide/generation for the same Y-chromosomal region from published human-chimpanzee comparisons depending on the generation and split times assumed.

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Capillary Sequence Traces of the Four Confirmed Mutations in Cell Line and Blood DNAsThe red arrowhead indicates the mutant position.
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fig1: Capillary Sequence Traces of the Four Confirmed Mutations in Cell Line and Blood DNAsThe red arrowhead indicates the mutant position.

Mentions: We next amplified the region spanning each candidate mutation from each of the cell lines used for chromosome sorting and sequenced them by conventional capillary sequencing. Twelve out of 18 (67%) first-class candidate mutations were confirmed in the cell-line DNA, and 0/5 (0%) second-class candidate mutations were confirmed. It therefore appears that the filtering criteria used were highly effective in identifying true mutations and that no more would be discovered if these criteria were relaxed. Nevertheless, mutations in cell-line DNA represent a combination of germline mutations carried by the donor and somatic mutations that have accumulated subsequently in culture [10, 11]. Blood DNA was available from both donors and from additional family members and was examined by capillary sequencing. Only four of the mutations were present in blood DNA (33%; Figure 1). Analysis of blood DNA from additional family members verified that three of the four mutations were also transmitted in the family and that the mutations had all occurred at different positions in the pedigree (Figure 2).


Human Y chromosome base-substitution mutation rate measured by direct sequencing in a deep-rooting pedigree.

Xue Y, Wang Q, Long Q, Ng BL, Swerdlow H, Burton J, Skuce C, Taylor R, Abdellah Z, Zhao Y, AsanMacArthur DG, Quail MA, Carter NP, Yang H, Tyler-Smith C - Curr. Biol. (2009)

Capillary Sequence Traces of the Four Confirmed Mutations in Cell Line and Blood DNAsThe red arrowhead indicates the mutant position.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748900&req=5

fig1: Capillary Sequence Traces of the Four Confirmed Mutations in Cell Line and Blood DNAsThe red arrowhead indicates the mutant position.
Mentions: We next amplified the region spanning each candidate mutation from each of the cell lines used for chromosome sorting and sequenced them by conventional capillary sequencing. Twelve out of 18 (67%) first-class candidate mutations were confirmed in the cell-line DNA, and 0/5 (0%) second-class candidate mutations were confirmed. It therefore appears that the filtering criteria used were highly effective in identifying true mutations and that no more would be discovered if these criteria were relaxed. Nevertheless, mutations in cell-line DNA represent a combination of germline mutations carried by the donor and somatic mutations that have accumulated subsequently in culture [10, 11]. Blood DNA was available from both donors and from additional family members and was examined by capillary sequencing. Only four of the mutations were present in blood DNA (33%; Figure 1). Analysis of blood DNA from additional family members verified that three of the four mutations were also transmitted in the family and that the mutations had all occurred at different positions in the pedigree (Figure 2).

Bottom Line: Candidate mutations were further examined by capillary sequencing in cell-line and blood DNA from the donors and additional family members.Twelve mutations were confirmed in approximately 10.15 Mb; eight of these had occurred in vitro and four in vivo.The latter could be placed in different positions on the pedigree and led to a mutation-rate measurement of 3.0 x 10(-8) mutations/nucleotide/generation (95% CI: 8.9 x 10(-9)-7.0 x 10(-8)), consistent with estimates of 2.3 x 10(-8)-6.3 x 10(-8) mutations/nucleotide/generation for the same Y-chromosomal region from published human-chimpanzee comparisons depending on the generation and split times assumed.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Sanger Institute, Hinxton, Cambs CB10 1SA, UK. ylx@sanger.ac.uk

ABSTRACT
Understanding the key process of human mutation is important for many aspects of medical genetics and human evolution. In the past, estimates of mutation rates have generally been inferred from phenotypic observations or comparisons of homologous sequences among closely related species. Here, we apply new sequencing technology to measure directly one mutation rate, that of base substitutions on the human Y chromosome. The Y chromosomes of two individuals separated by 13 generations were flow sorted and sequenced by Illumina (Solexa) paired-end sequencing to an average depth of 11x or 20x, respectively. Candidate mutations were further examined by capillary sequencing in cell-line and blood DNA from the donors and additional family members. Twelve mutations were confirmed in approximately 10.15 Mb; eight of these had occurred in vitro and four in vivo. The latter could be placed in different positions on the pedigree and led to a mutation-rate measurement of 3.0 x 10(-8) mutations/nucleotide/generation (95% CI: 8.9 x 10(-9)-7.0 x 10(-8)), consistent with estimates of 2.3 x 10(-8)-6.3 x 10(-8) mutations/nucleotide/generation for the same Y-chromosomal region from published human-chimpanzee comparisons depending on the generation and split times assumed.

Show MeSH