Additional file 2: Figure S2. of Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction

Quality of karyotype of JM8-derived clones is equivalent among distributing repositories. The figure shows the percentages of euploid clones sourced from the two main distributors of JM8-derived cells and GLT rate obtained with them. The numbers of clones in each instance is shown. These numbers illustrate that materials obtained from different distributors are of similar quality in terms of karyotype and GLT ability. Data was analysed using the Fisher Exact test that and showed no evidence of difference of quality between the two distributors. (PDF 77 kb

Additional file 6: Figure S5. of Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction

Outcome of chromosome counting by ddPCR with genomic DNA extracted from ear biopsies. The figure shows copy numbers obtained using the karyotype screen assay panel on DNA extracted from euploid ES cells (C), trisomic ES cells (Ts), X0 ES cells (Ns Y) and female mouse ear clip (E), using the same lysis method. Vertical bars are Standard Errors. The data illustrate that the assays are able detect the expected copy numbers on gDNA extracted from tisssues (2 Chr 1, 8 and 11) and that the number of these chromosomes is lower in genomes extracted from tissue cultures. (PDF 63 kb

Percentage of WT sequence recall with Fast basecalled data across a range of read depths.

This table summarises the percentage of WT sequence recall with Fast basecalled data across a range of depth of sequencing values with consensus thresholds ranging from 50% to 90%. (PDF)</p

Outcome of ONT sequencing of the <i>Mpeg1</i>-cre-80 founder animal visualised with IGV.

The alignment reflects the noisy nature of the method with errors distributed across the length of the sequenced segment. Note the complete alignment of reads (grey histograms) to designed mutant reference (sequence shown in zoomed in coloured frames). The dips in sequence depth (coloured frames) coincide with homopolymer repeats. (TIF)</p

Analysis of the G₁ generation containing animals interrogated by ONT sequencing for the <i>Inpp5k</i>-flox project.

The figure shows the PCR amplification of the genomic region of interest with (A) Inpp5k-F1 and Inpp5k-R1 primers (WT yields 1701 bp amplicon, floxed allele yields 1705 bp amplicon) and (B) LoxPF and LoxPR primers (floxed allele yields 1194 bp amplicon) from biopsies taken from the G1 animals derived from crossing founder animals Inpp5k-7 and Inpp5k-8 to WT. (C) The table details the G1 animals obtained from the two lines. The ID and outcome of PCR analysis of the region of interest, as well as the conclusion for each individual are shown. (D) The panels show the sequencing of PCR amplicon obtained from animal Inpp5k-7.1b with Inpp5k-F1 and LoxPR, and with LoxPF and Inpp5k-R1 respectively. (E) shows the sequencing of PCR amplicon obtained from animal Inpp5k-8.3d. Deviations from the intended mutant sequence are highlighted in blue. Animal(s) interrogated by ONT sequence analysis are highlighted in green. + is positive control amplified from an unrelated (A) WT, (B) floxed animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (TIF)</p

Percentage of WT sequence recall with HAC basecalled data across a range of Filtlong thresholds.

This table summarises the percentage of WT sequence recall across a range of Filtlong threshold values with consensus thresholds ranging from 50% to 100%. (PDF)</p

Additional file 20: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

The file contains the raw sequencing data obtained from the founders generated for the Rims1R655H point mutation. (ZIP 21747 kb

Analysis of the microinjection session containing animals interrogated by ONT sequencing for the <i>Tgfbr3</i> floxed project.

The figure shows the PCR amplification of the genomic region of interest with (A) Tgfbr3-F1 and Tgfbr3-R1 primers (WT yields 2339 bp amplicon, floxed yields 2443 bp amplicon) and (B) LoxPF and LoxPR primers (floxed yields 925 bp amplicon) from biopsies taken from the G0 animals. (C) The panels show the sequencing of PCR amplicons obtained from animal Tgfbr3-15 with Tgfbr3-F1 to LoxPR (to visualise 5’ loxP site) and LoxPF with Tgfbr3-R1 (to visualise 3’ loxP site). LoxP site sequences are highlighted in blue. (D) The table details the G0 animals analysed: The ID, outcome of PCR analysis of the region of interest and the conclusion for each individual are shown. Animal(s) interrogated by ONT sequence analysis are highlighted in green. + is positive control amplified from an unrelated (A) WT, (B) floxed animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (TIF)</p

<i>Mpeg1</i>-cre and <i>Cx3cl1</i>-flox allele.

The figure details the design of (A) the Mpeg1-cre and (B) the Cx3cl1-flox allele, respectively [12]. The positions of the primers used for analysis (sequences detailed in S7 Table) are shown, together with those of the ddPCR copy counting assays.</p

Additional file 18: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S16. Generation of a point mutation in Rims1 with ssODN donors. (a) The table details the F0 animals obtained for generation of Rims1 mutant with ssODN donors. The ID and outcome of sequencing the region of interest, as well as the conclusion for each individual are shown. (b) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from the F0 animals. Sequences of Rims1-ODN-151 mosaic and of sub-cloned amplicons are shown in Additional file 3: Figure S2u and v, demonstrating the presence of the desired mutation in this animal that was therefore mated. (c) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from Rims1-ODN-151’s offspring. Animal IDs are shown. + is positive control amplified from an unrelated WT animal. L1 = 1 kb DNA molecular weight (thick bands are 3 kb); L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). (d) The table details the first litter obtained by mating Rims1-ODN-151 with a WT mouse. The ID, outcome of sequencing the region of interest and copy counting of the region of interest as well as the conclusion for each individual are shown. Sequencing of Rims1-ODN-151.1g is shown in Additional file 3: Figure S2w and illustrates the failure of transmission of the desired allele. (PNG 893 kb