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

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

NB. No Sanger sequencing was performed on the Pam floxed G1 generation prior to analysis with ONT due to project timelines. The figure shows the PCR amplification of the genomic region of interest with (A) Pam-F1 and Pam-R1 primers (1431 bp amplicon) and (B) LoxPF and LoxPR primers (801 bp amplicon) from biopsies taken from Pam-3’s offspring. (C) The table details the first litter obtained by mating Pam-flox-3 with a WT mouse. The ID, outcome of PCR amplification of the regions of interest as well as the initial conclusion for each individual are shown. Sanger data obtained subsequent to the ONT run is displayed in (D) and (E) for animals Pam-3.1a and Pam-3.1b respectively. (D) The panels show the sequencing of PCR amplicon obtained from animal Pam-3.1a with Pam-F1 and Pam-R1. NHEJ events at each intended loxP insertion site are highlighted in blue, demonstrating that the LoxP product was generated by an off-target integration of the donor. (E) The panels show the sequencing of PCR amplicon obtained from animal Pam-3.1b with Pam-F1 and Pam-R1. LoxP insertion sites are highlighted in blue. However, when sequencing the LoxP PCR amplicons, more than one trace is present indicating multiple LoxP alleles and rearrangements. 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

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

ONT sequencing of <i>Cx3cl1</i>-flox conditional mutant project.

The figure summarises the outcome of ONT sequencing of founder animal Cx3cl1-flox-10 (A, B) and its offspring Cx3cl1-flox-10.1c (C, D) visualised with IGV. Panels A and C show alignments of all reads corresponding to the founder animal and his offspring, respectively. Panels B and D show alignments of only “Passed reads” (which are reads that have been filtered for the presence of a sequence that is specific to the mutant), providing a simplified readout of the presence of the desired mutation in each animal.</p

The sequences of sgRNAs, lssDNA templates, primers and probes employed in this study.

The sequences of sgRNAs, lssDNA templates, primers and probes employed in this study.</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

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