Number of false positive <i>de novo</i> mutations per billion bases detected by PolyMutt of jointly modeling for sequencing at coverage 5×–40× with Phred-scaled base quality Q20 (1% error rate) without mapping error in different pedigrees structures.

<p>Number of false positive <i>de novo</i> mutations per billion bases detected by PolyMutt of jointly modeling for sequencing at coverage 5×–40× with Phred-scaled base quality Q20 (1% error rate) without mapping error in different pedigrees structures.</p

Three-generation extended pedigrees.

<p>A) is a 3-generation extended pedigree with numbers labeling the individual heterozygous genotype mismatch rates (%) at coverage of 15× with base quality of Q20 without mapping error and panel B) labels the corresponding mismatch rates for the standard approach of ignoring relatedness. Panel C) and D) display the heterozygous mismatch rates (%) when a fixed sequencing effort of 150× is allocated differently to family members: Panel C) is for the situation where the founders are allocated 30× while non-founders have 5× and in Panel D) founders and non-founders have coverage of 6× and 21× respectively.</p

Genotype mismatch rates (%) for different family structures with sequencing coverage of 5×, 15×, and 30× and input bases with Phred-scaled quality Q20 (1% error rate) or Q30 (0.1% error rate) without mapping error.

<p>The mismatch rates are shown for 4 genotype categories: all genotypes (All), homozygous reference allele (HomRef), heterozygotes (Het), and homozygous alternative allele (HomAlt).</p

Mismatch rates (%) of 4 categories of genotypes by the reference allele frequencies for pedigrees of quartet (two siblings and their parents) with base quality Q20 at 15× without mapping error.

<p>The 4 categories are (A) overall genotypes, (B) homozygous alternative allele, (C) heterozygotes and (D) homozygous reference allele.</p

The receiver operating characteristic (ROC) curves of PolyMutt and the standard methods for <i>de novo</i> mutation (DNM) detection from empirically calibrated alignments of simulated reads with sequencing coverage of 30× with base quality of Q20.

<p>PolyMutt (ignoring relatedness) and GATK calls were obtained by jointly calling a trio assuming individuals in a trio are unrelated using Polymutt and GATK respectively.</p

Percentage of missing non-reference genotypes (i.e. false negatives) per individual in families for variants called by joint modeling family data and the standard approach of ignoring relatedness for sequencing coverage between 5× and 30× and for input sequence data with Phred-scaled quality of 20 (error rate of 1% per base) or 30 (error rate of 0.1% per base) without mapping error.

<p>For all scenarios 300 sequenced individuals were simulated.</p

Mendelian inconsistency rates per triplet (father, mother and offspring) for the genotypes by joint modeling of family data (top panel) and by the standard approach where the relatedness was ignored, i.e. individuals were treated as unrelated (bottom panel) for sequencing coverage of 5× to 30× and bases with Phred-scaled quality Q20 (1% error rate) and 30 (0.1% error rate) without mapping error.

<p>Mendelian inconsistency rates per triplet (father, mother and offspring) for the genotypes by joint modeling of family data (top panel) and by the standard approach where the relatedness was ignored, i.e. individuals were treated as unrelated (bottom panel) for sequencing coverage of 5× to 30× and bases with Phred-scaled quality Q20 (1% error rate) and 30 (0.1% error rate) without mapping error.</p

Power of detecting <i>de novo</i> mutations (DNM) in different pedigree structures for coverage from 5× to 40×.

<p>Panel A) shows the power for trios with base quality Q20 and Q30 and panel B) shows the power comparisons of trios, nuclear families with 2 and 3 siblings, and 3-generation extended pedigrees (shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002944#pgen-1002944-g001" target="_blank">Figure 1</a>) for base quality Q20 without mapping error.</p

Heterozygous mismatch rates (%) and Mendelian inconsistency rates (%) per site of call sets generated by PolyMutt (family-aware) and the standard approaches using PolyMutt (ignoring relatedness) and GATK from empirically calibrated alignments of simulated reads with base quality of Q20 in the pedigree shown in Figure 1.

<p>Heterozygous mismatch rates (%) and Mendelian inconsistency rates (%) per site of call sets generated by PolyMutt (family-aware) and the standard approaches using PolyMutt (ignoring relatedness) and GATK from empirically calibrated alignments of simulated reads with base quality of Q20 in the pedigree shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002944#pgen-1002944-g001" target="_blank">Figure 1</a>.</p

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