Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

<div><p>DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10^-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.</p></div

Correction: Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

<p>Correction: Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools</p

The sharing of mtDNA variants in parent-child trios.

<p>The sharing of mtDNA variants in parent-child trios.</p

The effect of age on the number of heteroplasmies in the unrelated SardiNIA sequencing project participants.

<p>The number of heteroplasmies increases with age with different (colored) minor allele fraction (MAF) thresholds. Each line plots the expected number of heteroplasmies based on the Poisson loglinear model against age at an MAF threshold; while the points represent the observed mean number of heteroplasmies in each age group (<40, 40–50, 50–60, 60–70, 70–80, >80).</p

Analysis pipeline to identify mtDNA variants includes aligning sequence reads to the whole genome reference (including mtDNA rCRS reference); extracting mtDNA reads; combining mapped and unmapped reads to do a second alignment to the shifted rCRS reference; applying the mtDNA variant caller separately to the reads mapped to the two linear mtDNA reference genomes; and combining the two sets of called mtDNA variants.

<p>Analysis pipeline to identify mtDNA variants includes aligning sequence reads to the whole genome reference (including mtDNA rCRS reference); extracting mtDNA reads; combining mapped and unmapped reads to do a second alignment to the shifted rCRS reference; applying the mtDNA variant caller separately to the reads mapped to the two linear mtDNA reference genomes; and combining the two sets of called mtDNA variants.</p

Two classes of base changes for homoplasmies and heteroplasmies.

<p>Two classes of base changes for homoplasmies and heteroplasmies.</p

Sharing of mtDNA variants among 2,077 SardiNIA sequencing project participants.

<p>Sharing of mtDNA variants among 2,077 SardiNIA sequencing project participants.</p

Population Genomic Analysis of Ancient and Modern Genomes Yields New Insights into the Genetic Ancestry of the Tyrolean Iceman and the Genetic Structure of Europe

<div><p>Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture.</p></div

Summary of datasets.

<p>*HG: Hunter-gatherer; F: Farmer.</p>†<p>only SNPs ascertained in non-European populations.</p

Allele sharing and D-tests with whole-genome datasets.

<p>(A) Normalized derived allele sharing rate of the Iceman with Eurasian whole genomes from Complete Genomics. Each circle represents the rate of sharing with a particular genome, grouped by population of origin. Positions on the y-axis have added jitter for ease of visualization. Populations with EUR suffix correspond to the European ancestry tracts of individuals of populations with known European admixture (ASW, MXL). Due to differences in admixture proportions among individuals from those populations, the total number of observations varies between individuals, indicated by the size of the circles. (B) D-test results for the ancient samples compared to populations from the 1000 Genomes project and Sardinia. Each panel shows results for a particular ancient sample, grouped by cultural context. Diamonds indicates the value of the D-statistic for a single D-test involving the ancient sample and a pair of modern populations, shown on the left and right of the panels. Significance at Z = 3 is indicated with filled diamonds, and the line shows the corresponding standard error of the D-statistic. Plot colors indicate different pairs of geographic regions within Europe (blue: Europe S/Europe N; green: Europe S/Europe S).</p