DOI: 10.5061/dryad.h18931zww
title: Reconstructing NOD-like receptor alleles with high internal conservation in Podospora anserina using long-read sequencing

description: NOD-like receptors (NLRs) are intracellular immune receptors that detect
 pathogen-associated cues and trigger defense mechanisms, including
 regulated cell death. In filamentous fungi, some NLRs mediate heterokaryon
 incompatibility, a self/non-self recognition process that prevents the
 vegetative fusion of genetically distinct individuals, reducing the risk
 of parasitism. The het-d and het-e NLRs in Podospora anserina are highly
 polymorphic incompatibility genes (het genes) whose products recognize
 different alleles of the het-c gene via a sensor domain composed of WD40
 repeats. These repeats display unusually high sequence identities
 maintained by concerted evolution. However, some sites within individual
 repeats are hypervariable and under diversifying selection. Despite
 extensive genetic studies, inconsistencies in the reported WD40 domain
 sequence have hindered functional and evolutionary analyses. Here we
 demonstrate that the WD40 domain can be accurately reconstructed from
 long-read sequencing (Oxford Nanopore and PacBio) data, but not from
 Illumina-based assemblies. Functional alleles are usually formed by 11
 highly conserved repeats, with different repeat combinations underlying
 the same phenotypic het-d and het-e incompatibility reactions. Protein
 structure models suggest that their WD40 domain folds into two 7-blade
 β-propellers composed of the highly conserved repeats, as well as three
 cryptic divergent repeats at the C-terminus. We additionally show that one
 particular het-e allele does not have an incompatibility reaction with
 common het-c alleles, despite being 11-repeats long. Our findings provide
 a robust foundation for future research into the molecular mechanisms and
 evolutionary dynamics of het NLRs, while also highlighting both the
 fragility and the flexibility of β-propellers as immune sensor domains.
description: This dataset consists of genome assemblies of three wildtype
 strains (Y+, Z+, and Wa63+) and nine lab strains that were the product of
 backcrossing
 different <em>het-e,</em> <em>het-d</em>, and
 <em>het-c</em> alleles into the genomic background of strains
 s ("little s"). Whole-genome DNA of most strains was extracted
 with the Zymo Quick-DNA Fungal/Bacterial Miniprep Kit D6005 (Zymo
 Research; https://zymoresearch.eu/). For the strain CmEm-, ~800mg of mycelia were used for high-molecular-weight DNA extraction using the QIAGEN Genomic-tip 100/G kit (Qiagen). Oxford Nanopore Technology (ONT) sequencing was performed in-house using a Native Barcoding Kit 24 V14 SQK-NBD114.24 and a MinION Mk1C machine following the standard protocol. In total, 12 strains were barcoded into two pools (pool1: CmEm-, CoEc+, CoEc-, Y+, Z+, and Wa63+ with barcodes 1 to 6, and pool2: CoEf+, ChEhDa+, ChEhDa-, CaDa-, CsDf+, and CsDf- with barcodes 7 to 12). Each pool was sequenced in two separate R10.4.1 flow cells (FLO-MIN114). Basecalling was performed using Dorado v. 0.5.3 (https://github.com/nanoporetech/dorado/) with the dna_r10.4.1_e8.2_400bps_sup@v4.3.0 model. The resulting BAM files were transformed into fastq files with the bam2fq program of SAMtools v. 1.19.2 (Danecek et al. 2021). Reads corresponding to the DNA Control Sample (DNA CS) introduced during library preparation were removed using chopper v. 0.7.0 (De Coster and Rademakers 2023). For each sample, we removed reads that contained perfect matches to ONT native barcodes assigned to other samples. We removed barcodes and performed minimum quality control with fastplong v. 0.2.2 (Chen 2023) and parameters --trimming_extension 20 -l 50 -q 15 -d 0.1 (hereafter, cleaned ONT reads). The cleaned ONT reads of each sample were used as input for Flye v. 2.9.3 (Kolmogorov et al. 2019), with parameters --nano-hq --iterations 2. In addition, the paired-end Illumina reads of the strains Wa63+, Y+, Z+, and Wa137- were retrieved from NCBI’s Sequence Read Archive (accession numbers SRX5458088, SRX5458091, SRX11405146, and SRX8537866) and assembled with SPAdes v. 4.0.0 (Prjibelski et al. 2020) using the --careful parameter and either the default k-mers setting (Wa63-, Z+, and Y-) or the k-mers 21, 33, 55, and 77 (all strains, "allkmers"). From all these assemblies, the nucleotide sequences of the <em>het-e,</em> <em>het-d</em>, and <em>het-r</em> genes were extracted and aligned manually (file 2024.09.10_hnwd_master_het_reAl_Illu_noGuides_noemptycols.fa).  Associated code can be found in the repository https://github.com/SLAment/FixingHetDE. New sequencing data was deposited in NCBI’s Sequence Read Archive Bioproject PRJNA1216259.  <strong>References</strong> Chen S. 2023. Ultrafast one‐pass FASTQ data preprocessing, quality control, and deduplication using fastp. <em>iMeta</em> 2: e107. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, et al. 2021. Twelve years of SAMtools and BCFtools. <em>GigaScience</em> 10: giab008. De Coster W, Rademakers R. 2023. NanoPack2: population-scale evaluation of long-read sequencing data. <em>Bioinformatics</em> 39: btad311. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. <em>Nat Biotechnol </em>37: 540–546. Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. 2020. Using SPAdes De Novo Assembler. <em>CP in Bioinformatics</em> 70: e102.
description: # Reconstructing NOD-like receptor alleles with high internal conservation
 in *Podospora anserina* using long-read sequencing
 [https://doi.org/10.5061/dryad.h18931zww](https://doi.org/10.5061/dryad.h18931zww) Principle Investigator Contact Information ``` Name: S. Lorena Ament-Velásquez Institution: Stockholm University ORCID: https://orcid.org/0000-0003-3371-9292 ``` ## Dataset Overview This dataset contains the whole genome assemblies required to replicate analyses in [Ament-Velásquez et al. (2025)](https://www.biorxiv.org/content/10.1101/2025.01.13.632504v1), where we show that the WD40 repetitive domain of the NOD-like receptors *het-e*, *het-d*, and *het-r* from the fungus *Podospora anserina* can be reconstructed from long-read sequencing (Oxford Nanopore Technology and PacBio) data, but not from Illumina-based assemblies. We sequenced the whole-genome of three wildtype strains (Y+, Z+, and Wa63+) and nine lab strains with **Oxford Nanopore Technology- ONT** (R10 flowcells). The lab strains were the product of backcrossing different *het-e*, *het-d*, and *het-c* alleles into the genomic background of strains s ("little s"). The backcrossed strains are designated by their reactive genotypes. For example, the strain CmEm- contains the *het-c* and *het-e* alleles of the French strain M, while having the non-reactive *het-d* allele of strain s. Likewise, the strain ChEhDa+ has the *het-c* and *het-e* alleles of the H strain and the *het-d* allele of the A strain. The exceptions are strains with a null *het-c* allele, here termed Co (CoEc+ and CoEf+). In addition, we re-assembled the **Illumina** reads of strains Wa63+, Y+, Z+, and Wa137-, which were retrieved from NCBI’s Sequence Read Archive (accession numbers SRX5458088, SRX5458091, SRX11405146, and SRX8537866). There are two versions of SPAdes assemblies (varying the k-mers used) for most strains, and only one for Wa137-. In addition, the nucleotide sequence alignment in fasta format of *het-e*, *het-d*, and *het-r* from all assemblies are found in `2024.09.10_hnwd_master_het_reAl_Illu_noGuides_noemptycols.fa`. The pipeline used to process this fasta file is available here: [https://github.com/SLAment/FixingHetDE](https://github.com/SLAment/FixingHetDE) All genome assemblies are provided in fasta format. ## Funding This work was supported by the Swedish Research Council (grant 2022-00341) and the Stiftelsen Anna-Greta och Holger Crafoords fond (CR2023-0039) to S.L.A.-V. ## Sharing/Access information This work is licensed under a CC0 1.0 Universal (CC0 1.0) Public Domain Dedication license. ### Related Data Sources We also analysed the whole genome assemblies from the following studies: * Vogan et al. (2019) "Combinations of Spok genes create multiple meiotic drivers in Podospora", eLife 8:e46454 [https://doi.org/10.7554/eLife.46454](https://doi.org/10.7554/eLife.46454), data available here: [https://datadryad.org/stash/dataset/doi:10.5061/dryad.vm1192g](https://datadryad.org/stash/dataset/doi:10.5061/dryad.vm1192g) * Vogan et al. (2021) "The Enterprise, a massive transposon carrying Spok meiotic drive genes", Genome Res. 31:789-798 [https://genome.cshlp.org/content/31/5/789](https://genome.cshlp.org/content/31/5/789), data available here: [https://datadryad.org/stash/dataset/doi:10.5061/dryad.4tmpg4f8m](https://datadryad.org/stash/dataset/doi:10.5061/dryad.4tmpg4f8m) * Ament-Velásquez (2024) "High-Quality Genome Assemblies of 4 Members of the Podospora anserina Species Complex", Genome Biology and Evolution, 16(3):evae034 [https://doi.org/10.1093/gbe/evae034](https://doi.org/10.1093/gbe/evae034), data available here: [https://datadryad.org/stash/dataset/doi:10.5061/dryad.1vhhmgr0j](https://datadryad.org/stash/dataset/doi:10.5061/dryad.1vhhmgr0j) ### Files and variables #### File: FixingHetDEdata\_v2.zip **Description:** This zip file contains all the genome assemblies from Illumina (folder `Illu_assemblies`) and ONT (`ONT_assemblies`) data, as well as the alignment of the *het* genes (`2024.09.10_hnwd_master_het_reAl_Illu_noGuides_noemptycols.fa`). All files are in fasta format. ## Code/software Code associated with this study can be found [here](https://github.com/SLAment/FixingHetDE). Since all files are in fasta format, they can be opened in any standard text editor, or in specialized software such as [AliView](https://ormbunkar.se/aliview/). ## Access information Illumina assemblies were derived from reads available at [NCBI’s Sequence Read Archive](https://www.ncbi.nlm.nih.gov/sra) (accession numbers SRX5458088, SRX5458091, SRX11405146, and SRX8537866). New sequencing data was deposited in NCBI’s Sequence Read Archive Bioproject PRJNA1216259.