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The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

Sanger sequencing validation for the inheritance of high level heteroplasmy SNV. Slide 1. CEPH family 1463 pedigree chart. Slide 2. Level of heteroplasmy calculated from the MitoRS data, and the Sanger sequencing data in both forward and reverse orientation. MitoRS calculated frequencies are highlighted in red (homoplasmy, > 98%) or orange (high frequency heteroplasmy, between 10% and 98%). The corresponding Sanger frequencies are highlighted in green for easier visualization. Slides 3 to 10. Chromatograms highlighting a difference between the two family members. The positions considered are shown with a black arrowhead. Slide 10. Virtual gel visualization (on a LabChip GX - Perkin Elmer) of the PCR amplification products analyzed by Sanger sequencing. (PPTX 513Â kb)

Overview of the MitoRS wet lab method. Total DNA (5 ng) is amplified by RCA. Sequencing libraries are subsequently generated using the Nextera XT kit from Illumina, starting from 1 ng amplification product. Libraries are pooled at equimolar ratios and sequenced on a HiSeq 2500 (Illumina) using rapid mode for a paired-end run of 2 x 150 cycles. (PPTX 40 kb)

The RCA procedure is robust. A. RCA does not introduce technical variability. Each individual plasmid DNA was run as four independent replicates for both conditions (with or without RCA). The technical reproducibility of the variant frequency call was evaluated by calculating the standard deviation within the four replicates. Value are plotted for each individual position of the reference sequence, for the two plasmids sequenced. Data are directly extracted from mpileup analysis applying a VarScan p-value threshold of 0.001. Left panels: plasmid 1, right panels: plasmid 2, top panels: crude plasmid DNA (no RCA amplification), bottom panels: RCA amplified plasmid DNA. B. Origin of the nonspecific RCA products. The sequences surrounding the unique position for which a large difference was observed between the crude plasmid DNAs and the RCA products were aligned. The sequence obtained from contaminating NTC reads is shown. The common (between the two plasmids) non-accurate position is boxed (“G” in the NTC, “A” in the two plasmids). This region is part of the plasmid DNA origin of replication. (PPTX 178 kb)

Overview of the MitoRS analysis methods. FastQ files are aligned with BWA to the original, and an origin-shifted version, of the DNA reference sequence (refer to Additional file 2 for details). Variant frequency is evaluated by samtools mpileup. Low frequency variants are finally identified using VarScan 2. Datasets generated from both the original reference and the shifted reference are merged, keeping only the per position values from the dataset with the highest coverage. Data are further processed to generate 1) a csv file summarizing the sequencing results observed at each individual mitochondrial DNA position and 2) a fastA file representing the corresponding consensus sequence. More details can be found in the Methods section. (PPTX 264Â kb)

RCA enriches circular versus linear templates. A. Principle of circular DNA enrichment. A single priming event will generate several concatenated copies of a circular template. At the opposite, a single copy will be amplified if the template DNA is linear. B. The RCA amplified material is mostly mtDNA. Digestion of the mouse DNA RCA product with the SpeI restriction endonuclease results in the expected mtDNA digestion product with only low amount of undigested DNA left. SpeI restriction digest is expected to result in four fragments for the B6D2F1 strain (7’398, 3’759, 3’105, and 2’035 bp) and five fragments for the NMRI strain (7’398, 3’105, 2’150, 2’037, and 1609 bp). Ladder sizes imprecision is in accordance with the Agilent Genomic DNA ScreenTape specifications. (PPTX 133 kb)

Mouse SNV frequencies are homogenous within the 88 position analyzed. A. Individual SNV frequency. For the 12 mouse mtDNA mixture ratios tested, the frequencies measured for each SNV (88 in total) were plotted and summarized as a boxplot. The boxplot whiskers highlight the extreme values (min and max). Note that the scale is different for each plot. The eight positions showing the highest frequency underestimation are highlighted in blue. These data were used to build the Fig. 4. The raw data are available from the Additional file 7. B. The eight SNV with underestimated frequency are located into two dense clusters. For each 88 SNV, the deviation from the theoretical frequency was calculated and plotted versus their position in the mitochondrial genome. The eight positions showing a systematic frequency underestimation highlighted in A. are also shown in blue. They cluster into two very short genomic regions. (PPTX 484 kb)

La qualité pour agir est l'une des conditions de recevabilité de la requête individuelle (article 34 de la Convention), Depuis une vingtaine d'années, elle fait l'objet d'une jurisprudence de plus en plus abondante. La thèse propose un exposé systématique de cas d'espèce et l'analyse critique des solutions apportées par la Cour. Elle présente, en particulier s'agissant des notions de victime directe, indirecte et potentielle, des solutions visant à renforcer la cohérence et la prévisibilité de la jurisprudence, notamment par la fixation de critères clairs. D'une manière générale, la Cour devrait s'appuyer sur le principe de subsidiarité pour responsabiliser davantage les Etats parties. Son interprétation de la notion de victime devrait inviter les Etats à mettre en place des voies de recours internes effectives et les requérants à obtenir réparation devant les juridictions nationales.

Box plots of CSF metabolite measurements for CDR of 0 and CDR >0 (i.e., CDR 0.5 or 1). Level of significance is indicated as p value after Bonferroni correction. Values were log-transformed. Figure S2. Box plots of plasma metabolite measurements for CDR 0 and CDR >0 (i.e., CDR 0.5 or 1). Level of significance is indicated as p value after Bonferroni correction. Concentration values were log-transformed. Figure S3. CSF and plasma metabolites correlated with CSF p-tau181 (a) and CSF tau (b) in APOE ε4 carriers (n = 37). Concentration values were log-transformed. Each dot represents a subject. The black line represents the linear fit, and the gray shading represents its confidence interval. Figure S4. Box plots of plasma metabolite measurements in APOE ε4 carriers (n = 37) according to CSF p-tau181/Aβ1–42 ratio (i.e., “low” when p-tau181/Aβ1–42 is ≤0.0779 and “high” when p-tau181/Aβ1–42 is > 0.0779) for negative and positive CSF profiles of AD pathology, respectively. Concentration values were log10-transformed. Level of significance is indicated as p value after Bonferroni correction. Supplementary Methods. (PDF 831 kb)

Haplogroup determination for the CEPH family 1463. The fastA file generated for each individual of the CEPH family 1463 was submitted to the Haplofind tool. Note that N are considered as deletion. When the completion status is “No”, Haplofind was not able to determine the exact subhaplogroup. (DOCX 39 kb)

Supporting Information. (DOCX 56Â kb)