A framework for the detection of de novo mutations in family-based sequencing data

Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father's age at conception and the number of DNMs in female offspring's X chromosome, consistent with previous literature reports

Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-bgd in the endoplasmic reticulum

The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1 alpha, EF-1 beta, EF-1 gamma and EF-1 delta which induce efficient transfer of aminoacyl-tRNA to the ribosome, In this process EF-1 alpha . GTP acts as the carrier of the aminoacyl-tRNA on its way to the ribosome, After release of aminoacyl-tRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1 alpha . GDP form is recycled to EF-1 alpha . GTP by EF-1 beta gamma delta, In eukaryotic cells the concentration of EF-1 alpha exceeds that of the complex beta gamma delta by a factor of 5-10.In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed.In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1 beta gamma delta, was found to co-localize with the endoplasmic reticulum (ER), displaying a distinct fine-structure in its staining pattern, The guanine nucleotide-binding subunit of EF-1, EF-1 alpha, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1 alpha, EF-1 beta, EF-1 gamma and EF-1 delta which induce efficient transfer of aminoacyl-tRNA to the ribosome, In this process EF-1 alpha . GTP acts as the carrier of the aminoacyl-tRNA on its way to the ribosome, After release of aminoacyl-tRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1 alpha . GDP form is recycled to EF-1 alpha . GTP by EF-1 beta gamma delta, In eukaryotic cells the concentration of EF-1 alpha exceeds that of the complex beta gamma delta by a factor of 5-10.In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed.In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1 beta gamma delta, was found to co-localize with the endoplasmic reticulum (ER), displaying a distinct fine-structure in its staining pattern, The guanine nucleotide-binding subunit of EF-1, EF-1 alpha, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.A