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

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

The sharing of mtDNA variants in parent-child trios.

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

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

Signature Genes for Specific Groups of Early Embryos and Stem Cells

<p>Signature Genes for Specific Groups of Early Embryos and Stem Cells</p

Examples of NIA-Only cDNA Clones and RT–PCR Results

<p>Expression pattern of 19 novel cDNA clones in 16 different cell lines or tissues: unfertilized egg, E3.5 blastocyst, E7.5 whole embryo (embryo plus placenta), E12.5 male mesonephros (gonad plus mesonephros), newborn brain, newborn ovary, newborn kidney, embryonic germ (EG) cell, embryonic stem (ES) cell (maintained as undifferentiated in the presence of LIF), trophoblast stem (TS) cell, mesenchymal stem (MS) cell, osteoblast, neural stem/progenitor (NS) cell, NS differentiated (differentiated neural stem/progenitor cells), and hematopoietic stem/progenitor (HS) cells. Glyceraldegyde-3-phosphate dehydrogenase (GAP-DH) was used as a control. A U number is assigned to each gene in the gene index (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000074#sd002" target="_blank">Dataset S2</a>). The exon number was predicted from alignment with the mouse genome sequence, and the amino acid sequence was predicted with the ORF finder from NCBI.</p

PCA Analysis of EST Frequency

<p>The results were obtained by analyzing 2,812 genes that exceeded 0.1% in at least one library. (A) 3D biplot that shows both cell types (red spheres) and genes (yellow boxes). (B) 2D PCA of cell types. EST frequencies were log-transformed before the analysis. Names of some cells and tissues are abbreviated as follows: 6.5 EP, E6.5 whole embryo (embryo plus placenta); 7.5 EP, E7.5 whole embryo (embryo plus placenta); 8.5 EP, E8.5 whole embryo (embryo plus placenta); 9.5 EP, E9.5 whole embryo (embryo plus placenta); 7.5 E, E7.5 embryonic part only; 7.5 P, E7.5 extraembryonic part only; NbOvary, newborn ovary; NbBrain, newborn brain; NbHeart, newborn heart; NbKidney, newborn kidney; 13.5 VMB, E13.5 ventral midbrain dopamine cells; 12.5 Gonad (F), E12.5 female gonad/mesonephros; 12.5 Gonad (M), E12.5 male gonad/mesonephros; HS (Kit⁻, Sca1⁻), hematopoietic stem/progenitor cells (Lin⁻, Kit⁻, Sca1⁻); HS (Kit⁻, Sca1⁺), hematopoietic stem/progenitor cells (Lin⁻, Kit⁻, Sca1⁺); HS (Kit⁺, Sca1⁻), hematopoietic stem/progenitor cells (Lin⁻, Kit⁺, Sca1⁻); HS (Kit⁺, Sca1⁺), hematopoietic stem/progenitor cells (Lin⁻, Kit⁺, Sca1⁺); and NS-D, differentiated NS cells.</p