Journal: medRxiv

Article Title: Gene-Pseudogene Inversions as a Hidden Source of Missing Heritability

doi: 10.1101/2025.10.01.25336578

Figure Lengend Snippet:

Article Snippet: Sequencing adaptors and indexes were added to DNA fragments using NEBNext Multiplex Oligos for Illumina kit (E7335 and E7500, New England BioLabs) and NEBNext Ultra II Q5 Master Mix (M0544, New England BioLabs).

Techniques: Sequencing, Disruption

Journal: medRxiv

Article Title: Gene-Pseudogene Inversions as a Hidden Source of Missing Heritability

doi: 10.1101/2025.10.01.25336578

Figure Lengend Snippet: Identification of SORD/SORD2P inversion as a common pathogenic allele in CMT-SORD. a) IGV visualization of the inversion breakpoint (intron 5 SORD2P and intron 4 SORD). Split and soft-clipped long-reads (red arrows) identify insertions/deletions of Alu elements specific to SORD or SORD2P, thus delineating the genomic interval of the breakpoints of the inversion between SORD and SORD2P (black boxes). Short-read sequencing fails to resolve the inversion due to high sequence homology. b) Schematic representation of the inversion’s genomic effect: the first four exons of SORD are replaced by the first five exons of SORD2P, which harbor multiple nonsense variants, leading to non-sense mediated decay and loss of SORD function. c) Optical Genome Mapping (OGM) of the inversion. The first breakpoint is located within the SORD2P pseudogene, while the second is within SORD. d) Zoom-in of the Hi-C ∼1 Mb region surrounding the SORD-SORD2P locus (skin fibroblasts; hg19; 5 kb resolution; raw counts) in a control (panel A; CTR) and the patient (panel B; proband N, SORD-CM). Statistically significant chromatin contacts identified by FitHiC2 are shown for both the control and patient samples. A patient-specific interaction (dark red curve, panel B) between chromosome 15 positions 45,132,499 and 45,352,501 was detected, consistent with the SORD/SORD2P inversion identified in the patient. Panel C displays a heatmap obtained with the subtraction method (SB map; patient - control), in which the inversion is visualized as a characteristic bow-tie pattern located distant from the matrix (arrows). e) Schematic representation of the breakpoint-specific PCR assay (top) and corresponding gel electrophoresis of PCR products from SORD-CMT patients and controls. Primers target divergent Alu elements in SORD2P intron 6 and SORD intron 5. In the reference genome, primers F1 and F2 are oriented in the same direction and ∼226 kb apart, precluding amplification. Upon inversion, F1 now lies ∼3.2 kb from F2 and in opposite direction, thus enabling amplification of a PCR product. Gel electrophoresis of breakpoint PCR assay shows the presence of a 3.2 Kb PCR product in probands P1 and P2 (heterozygous for SORD c.757delG and the inversion), but not in control (C1) or in probands that do not carry the inversion (S1–S3). f) Pie chart depicting the prevalence of SORD variants in a cohort of 156 CMT-SORD patients. SORD/SORD2P inversions represent the third most common pathogenic allele.

Article Snippet: Sequencing adaptors and indexes were added to DNA fragments using NEBNext Multiplex Oligos for Illumina kit (E7335 and E7500, New England BioLabs) and NEBNext Ultra II Q5 Master Mix (M0544, New England BioLabs).

Techniques: Sequencing, Hi-C, Control, Nucleic Acid Electrophoresis, Amplification

Journal: bioRxiv

Article Title: Polycomb repression works without Siesta

doi: 10.1101/2025.07.18.664654

Figure Lengend Snippet: A. To verify the presence of Sce alleles, genomic DNA from flies of the indicated genotypes was used as a template for PCR using the primer combination shown in the table. DNA from Oregon-R strain was used as a control. PCR products were analysed by gel electrophoresis in 1% agarose gel along with the Gene Ruler 1kb Plus molecular weight marker (M). B. Chromatograms of sequencing reactions with PCR products from ( A ) show the Isoleucine to Alanine substitution at position 48 of the transgenic Sce .

Article Snippet: 1μl of the resulting product from the second PCR round was used in the third round of PCR to incorporate Illumina sequencing adaptors.

Techniques: Control, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Sequencing, Transgenic Assay

Journal: bioRxiv

Article Title: Polycomb repression works without Siesta

doi: 10.1101/2025.07.18.664654

Figure Lengend Snippet: A. Schematics of a gene to be edited with locations of target sequences for gRNAs and primers for genotyping. gRNA_A1 and gRNA_A2 are used for the first round of editing, gRNA_B1 and gRNA_B2 are used for the second round of editing. Primer_F1 and Primer_R1 are used for amplification of the junction after DNA repair and sequencing. Primer_F2 and Primer_R2 are used to screen for the clones with homozygous deletion; no PCR product is expected in homozygous cells. B. The schematic of unedited alleles. Rectangles correspond to the position of target sequences for gRNAs, vertical lines inside each rectangle indicate the position of the Cas9 cut in case of the precise deletion. C. Combination of alleles in heterozygous cells after the first round of editing. Most of the selected single cell clones bear only one allele with a deletion. D. The two most frequent combinations of alleles in the resulting clones after the second round of editing.

Article Snippet: 1μl of the resulting product from the second PCR round was used in the third round of PCR to incorporate Illumina sequencing adaptors.

Techniques: Amplification, Sequencing, Clone Assay

Journal: bioRxiv

Article Title: Polycomb repression works without Siesta

doi: 10.1101/2025.07.18.664654

Figure Lengend Snippet: A. The Sce gene structure and location of target nucleotide sequences for guide RNAs and primers. The Sce gene, mRNA and cDNA are represented as long arrows. Primer locations are indicated as pink arrows, locations of target nucleotide sequences for guide RNAs with corresponding Protospacer Adjacent Motifs (PAMs) are shown as grey boxes. The nucleotide sequences of PCR primers are listed in Table S3. B. DNA sequences of both homologous chromosomes at the editing site in unedited cells, heterozygous cells and homozygous cells from the resulting cell lines. The gRNA target sequences are underlined and shown in green for the outer pair of gRNAs, in blue for the inner pair of gRNAs. Triplets in bold correspond to PAMs. Red dashed lines mark the position of deleted nucleotides. Blue and grey vertical shadows on sequencing chromatograms mark the position of the junction.

Article Snippet: 1μl of the resulting product from the second PCR round was used in the third round of PCR to incorporate Illumina sequencing adaptors.

Techniques: Sequencing