Pervasive influence of repeat-induced point mutations on single-copy functional genes confirm its importance in the evolution of Neurospora crassa

FGSC2225 Genome Assembly FGSC2225-Genome.tar.gz provides the genome assembly of Neurospora crassa, including the complete genome sequence, annotated CDS sequences, and predicted peptide sequences. Sanger verification data Methods To address potential mapping artifacts and short-read misalignment associated with self-repetitive sequences, we conducted a pairwise BLAST analysis of all coding sequences within the FGSC2489 reference genome. A lenient threshold (E-value < 1e-4 and alignment length > 80 bp) was utilized to identify regions of internal homology, resulting in the identification of 132 HRAGs. To experimentally validate the accuracy of the bioinformatically predicted variants, 8 HRAGs harboring candidate SNPs were selected for further PCR amplification. Primers were designed using Primer 5 (detailed in Sanger_verification_summary_tables.xlsx), and the resulting amplicons were subjected to Sanger sequencing to verify the identified variants. Additionally, to rigorously evaluate the accuracy of novel SNP identification within internal homology domains, the sequence of the Chitinase-1 gene was amplified and verified by Sanger sequencing across both parental strains and three representative offspring. Results To exclude potential mapping artifacts, we selected a subset of HRAG loci that are more susceptible to such issues for experimental validation, including Chitinase-1, which contains short internal repetitive elements and exhibits a high local density of mutations. Such features can increase the likelihood of ambiguous short-read alignment or clustering of apparent SNPs. These loci were therefore validated using Sanger sequencing. All tested variants were fully consistent with short-read–based variant calls (Sanger_verification_summary_tables.xlsx), confirming the robustness of our variant detection. The complete data matrix summarizing these PCR validation experiments and Sanger sequencing results is provided in Sanger_verification_summary_tables.xlsx. Sanger_verification_data.zip contains supplementary PCR validation results, demonstrating that the substantial observed mutations at coding genes in N. crassa are not artifacts of sequence repetition or read misalignment, but represent genuine, reproducible genetic variation. Folder structure ---------------- Sanger_verification_data/ ├── ab1/ ├── ref_fasta/ ├── screenshot/ └── readme.txt Contents -------- 1. ab1/ This folder contains the original Sanger sequencing chromatogram files in AB1 format. These files can be opened with software such as SnapGene Viewer, Chromas, FinchTV, or other chromatogram viewers. They are used to inspect peak quality and manually confirm Sanger validation results. 2. ref_fasta/ This folder contains FASTA files used for sequence alignment. They can be used to verify whether the detected variant is supported by the sequencing result. 3. screenshot/ This folder contains screenshots of chromatograms, sequence alignments, or validation results. These images are provided for quick visual inspection and documentation. Visualization of these files was implemented using SnapGene. 4. readme.txt This file describes the organization and purpose of the supplementary materials. Tips ------------- Detailed information on the corresponding PCR regions and SNP sites is provided in Supplemental Datasheet 4. The recommended workflow is: 1. Open the AB1 chromatogram files in the ab1 folder to check raw Sanger sequencing quality. 2. Compare the corresponding sequences with the files in alignment_fasta. 3. Refer to the images in screenshot for visual confirmation of the Sanger validation result.