Bacteriophage lambda

All DNA samples were mixed in TBE buffer (Medicago, 10 x TBE tablets) diluted in milli-Q water to the desired ionic strength. The oxygen scavenger β-mercaptoethanol (Sigma-Aldrich) was added to the buffer (3% v/v) to suppress photonicking of the DNA. YOYO-1 was purchased from Invitrogen and DNA from phage lambda (λ-DNA, 48.5 kb) was purchased from New England Biolabs.
The nanofluidic chips were fabricated in fused silica according to methods described elsewhere (6 (link)), with a cross-section of ∼100 × 150 nm² and a length of ∼500 µm. All experiments were conducted in a nanofluidic chip consisting of pairs of microchannels that are spanned by nanochannels. The DNA sample solution was loaded into one of the microchannels of the chip and transferred to the nanochannel array by pressure. By applying pressure over two connected microchannels, the DNA was subsequently injected into the nanochannels. The DNA was imaged using an epi-fluorescence microscope (Zeiss AxioObserver.Z1) equipped with a Photometrics Evolve EMCCD camera and a 100× oil immersion TIRF objective (NA = 1.46) from Zeiss. Stacks of 200 images were recorded for each molecule, using the AxioVision software, with an exposure time of 100 ms per image at maximum speed, corresponding to approximately seven frames per second. Data analysis was performed with the freeware ImageJ (www.imagej.com) and a custom-written MatLab based software. For each molecule, kymographs (timetraces) were first extracted. Subsequently, each line in the kymograph—corresponding to one frame in the original time series—was fitted with a convolution of a box function and an error function (6 (link)). This gives the position and extension of the molecule in each frame that is used to align the kymograph as well as obtaining average intensities and fluctuations.
The fluorescence intensity was normalized to a distinct upper limit in fluorescence intensity identified from measurements. This upper limit is assumed to correspond to the fluorescence intensity of fully intercalated DNA, ∼1 YOYO molecule every 4 bp (21 (link),22 (link)). Extrapolation of the extension of native DNA was obtained by a linear fit of the first eight binned values for each ionic strength. The straight lines in Figure 3B were calculated from the extrapolated extension of the native DNA, again assuming a maximal intercalation of one YOYO every 4 bp and adding the resulting increase in contour length of 0.51 nm per YOYO molecule (22 (link)), taking the relative extension of the native DNA compared with the full contour length into account.

Nanopore sequence data was filtered to remove the control lambda-phage and sequences shorter than 1,000 bases using the nanopack tool suite [v1.0.1] (De Coster et al. 2018 (link)). Trimmomatic [v0.32] (Bolger et al. 2014 (link)) was used to remove adapters, trim low-quality bases, and filter out reads shorter than 85 bp. The filtered nanopore data were assembled into contigs using wtdbg2 [v2.4] (Ruan and Li 2020 (link)). The contigs were polished using two iterations of racon [v1.4.0] (Vaser et al. 2017 (link)) with minimap2 [v2.17] (Li 2018 (link)) mapping the nanopore reads. The contigs were further polished with Illumina paired-end read data using pilon [v1.23] (Walker et al. 2014 (link)) with bwa [v0.7.10] (Li 2013 ) mapping the Illumina paired reads. The resulting contigs were scaffolded using Bionano Solve [Solve3.4.1_09262019] using the optical mapping data generated from the Saphyr run. SALSA [v2.3] (Ghurye et al. 2019 (link)) was used to produce super-scaffolds using the Hi-C library and the Bionano scaffolded sequences. Those scaffolds larger than 10Mb were linked and oriented based on the Onychostoma macrolepis genome (Sun et al. 2020 (link)), the chromosome assembly most similar to L. rohita available on NCBI, using RagTag [v1.1.1] (Alonge et al. 2022 (link)).
RepeatModeler [v2.0.1] (Flynn et al. 2020 (link)) and RepeatMasker [v4.1.1] (Smit et al. 2013 ) were used to create a species-specific repeat database, and this database was subsequently used by RepeatMasker to mask those repeats in the genome. All available RNA-seq libraries for L. rohita (comprising brain, pituitary, gonad, liver, pooled, and whole body tissues for both sexes; Supplementary Table 1) were downloaded from NCBI and mapped to the masked genome using hisat2 [v2.1.0] (Kim et al. 2019 (link)). These alignments were used in both the mikado [v2.0rc2] (Venturini et al. 2018 (link)) and braker2 [v2.1.5] (Brůna et al. 2021 (link)) pipelines. Mikado uses putative transcripts assembled from the RNA-seq alignments generated via stringtie [v2.1.2] (Kovaka et al. 2019 (link)), cufflinks [v2.2.1] (Trapnell et al. 2012 (link)), and trinity [v2.11.0] (Grabherr et al. 2011 (link)) along with the junction site prediction from portcullis [v1.2.2] (Mapleson et al. 2018 (link)), the alignments of the putative transcripts with UniprotKB Swiss-Prot [v2021.03] (The UniProt Consortium 2021 (link)), and the ORFs from prodigal [v2.6.3] (Hyatt et al. 2010 (link)) to select the best representative transcript for each locus. Braker2 uses those RNA-seq alignments and the gene prediction from GeneMark-ES [v4.61] (Borodovsky and Lomsadze 2011 (link)) to train a species-specific Augustus [v3.3.3] (Stanke et al. 2006 (link)) model. Maker2 [v2.31.10] (Holt and Yandell 2011 (link)) predicts genes based on the new Augustus, GeneMark, and SNAP models derived from Braker2 along with the Mikado predicted transcripts as an external ab-initio source, modifying the predictions based on the available RNA and protein evidence from the Cyprinidae family in the NCBI RefSeq database. Any predicted genes with an annotation edit distance (AED) above 0.47 were removed from further analysis. The remaining genes were functionally annotated using InterProScan [v5.47-82.0] (Jones et al. 2014 (link)) and BLAST + [v2.9.0] (Camacho et al. 2009 (link)) alignments against the UniprotKB Swiss-Prot database. BUSCO [v5.2.2] (Manni et al. 2021 (link)) was used to verify the completeness of both the genome and annotations against the actinopterygii_odb10 database. Lastly, genes spanning large gaps or completely contained within another gene on the opposite strand were removed using a custom Perl script (https://github.com/IGBB/rohu-genome/).

High-molecular-weight DNA extracted from all enrichment cultures was combined in equimolar amounts and used to prepare two metagenomic fosmid libraries, IS_Lib1 (Cavascura enrichments) and IS_Lib2 (Maronti enrichments) using the CopyControl fosmid library pCC2FOS production kit (Epicentre Technologies, Madison, WI, USA). DNA was end-repaired to generate blunt-ended 5′-phosphorylated fragments according to the manufacturer’s instructions. Subsequently, DNA fragments in the range of 30 to 40 kbp were resolved by gel electrophoresis (2 V cm⁻¹ overnight at 4°C) and recovered from 1% low-melting-point agarose gel using GELase 50× buffer and GELase enzyme (Epicentre). Nucleic acid fragments were then ligated to the linearized CopyControl pCC2FOS vector following the manufacturer’s instructions. After the in vitro packaging into the phage lambda (MaxPlax lambda packaging extract, Epicentre), the transfected phage T1-resistant EPI300-T1^RE. coli cells were spread on Luria-Bertani (LB) agar medium containing 12.5 μg mL⁻¹ chloramphenicol and incubated at 37°C overnight to determine the titer of the phage particles. The resulting libraries had estimated titers of 14 × 10⁴ and 1 × 10⁴ nonredundant fosmid clones in the IS_Lib1 and IS_Lib2 libraries, respectively. For long-term storage, E. coli colonies were washed from the agar surface using liquid LB medium containing 20% (vol/vol) sterile glycerol, and the aliquots were stored at −80°C.

For metaphase spread preparation, mitoses were mechanically collected by blowing the medium on the dish surface. Chromosome preparations were performed with the standard air-drying procedure and were used for karyotype analysis or fluorescence in situ hybridization (FISH).
Whole genomic DNA from Przewalski’s horse fibroblasts was extracted according to standard procedures. BAC clones (CHOR1241-17G8, coordinates in EquCab2.0: chr5:37,011,456–37,167,308; CHOR1241-25H7 coordinates in EquCab2.0: chr5: 98,031,303–98,224,666) and lambda phage 37cen and 2PI clones [16 (link),53 (link)] were extracted from 10 mL bacterial cultures with the Quantum Prep Plasmid miniprep kit (BioRad, Milan, Italy), according to supplier instructions. All probes were labeled by nick translation with Cy3-dUTP (Enzo Life Sciences, Milan, Italy) or Alexa488-dUTP (Life Technologies, Monza, Italy) as previously described [64 (link)]. FISH was performed as previously described [68 (link)].
Chromosomes were counterstained by DAPI. Digital grayscale images for fluorescence signals were acquired with a fluorescence microscope (Zeiss Axio Scope.A1, Zeiss, Göttingen, Germany) equipped with a cooled CCD camera (Teledyne Photometrics, Birmingham, UK). Pseudocoloring and merging of images were performed using the IPLab 3.5.5 Imaging Software (Scanalytics Inc., Fairfax, VA, USA). Chromosomes were identified by DAPI banding according to the published karyotypes [49 (link),50 (link)].