DOI: 10.48802/owey.ktbvjzxn
title: Genomic DNA and Hi-C sequencing of 14 representative bacterial strains with large extrachromosomal replicons

description: This dataset was generated to investigate how large extrachromosomal replicons (ERs) integrate into bacterial replication and segregation systems. These ERs, sometimes nearly the size of the main chromosome, are widespread among bacteria, yet their behavior during the cell cycle remains poorly understood. The goal of this study was to explore their replication timing and spatial organization within the cell.

The dataset contains paired-end FastQ files from genomic DNA and Hi-C sequencing of 14 bacterial strains harboring large ERs. Strains include representatives from Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deinococci, and Spirochaetes.

The dataset is organized into one folder per bacterial strain. Each folder contains:

 (1) FastQ files from Hi-C sequencing. Hi-C is a chromosome conformation capture technique performed on exponentially growing cells. It reveals physical interactions between different genomic regions, including contacts between chromosomes and ERs, providing insights into their spatial organization.

(2) FastQ files from genomic DNA sequencing. Genomic DNA was extracted from exponential and stationary phase cultures and used for Marker Frequency Analysis (MFA). MFA estimates replication dynamics by analyzing read coverage along the genome, enabling identification of replication origins and termini, as well as estimation of replication fork speed and the relative timing of initiation and termination between replicons.
description: * Strains
Allorhizobium vitis S4 (formerly Agrobacterium vitis, NCBI:txid311402)
Brucella anthropi ATCC 49188 (formerly Ochrobactrum anthropi, NCBI:txid439375)
Burkholderia cenocepacia J2315 (NCBI:txid216591)
Burkholderia cepacia ATCC 25416 (NCBI:txid983594)
Cereibacter sphaeroides 2.4.1 (formerly Rhodobacter sphaeroides, NCBI:txid272943)
Cupriavidus necator N-1 (NCBI:txid1042878)
Deinococcus radiodurans R1(NCBI:txid243230)
Leptospira biflexa serovar Patoc strain 'Patoc 1 (Paris)' (NCBI:txid456481)
Paracoccus aminophilus JCM 7686(NCBI:txid1367847)
Paracoccus aminovorans JCM 7685 (CIP 106076)
Paracoccus denitrificans PD1222 (NCBI:txid318586)
Pseudoalteromonas translucida TAC125 (formerly Pseudoalteromonas haloplanktis, NCBI:txid326442)
Sinorhizobium meliloti 1021 (NCBI:txid266834).

* Growth conditions
All strains, except A. vitis, P. translucida, D. radiodurans, L. biflexa, and L. interrogans, were grown in LB Lennox medium at 30°C. A. vitis was grown in TY medium (bacto-tryptone 5 g/L, yeast extract 3 g/L, CaCl₂·2H₂O 0.87 g/L) at 30°C, P. translucida in TYP medium (bacto-tryptone 16 g/L, yeast extract 16 g/L, NaCl 20 g/L) at 20°C, D. radiodurans in TSB medium (Tryptic Soy Broth) at 30°C, and Leptospira biflexa in EMJH (Ellinghausen-McCullough-Johnson-Harris) medium at 30°C.

* MFA
Overnight cultures were initiated from fresh streaks on solid medium by inoculating 3 mL of liquid medium and incubating for 24 hours with shaking (150 rpm). To obtain exponential phase cultures, 0.1 mL of the overnight culture was transferred into 100 mL of fresh medium and incubated in a shaking water bath (180 rpm) until reaching an OD₄₅₀ of 0.2–0.4, except for L. biflexa, which was grown to an OD₄₂₀ of 0.2. For exponential phase samples, bacteria were harvested by centrifuging the total culture volume in two 50 mL Falcon tubes at 4°C for 10 minutes at 8000 rpm. The supernatant was discarded, and the bacterial pellets were immediately stored at -20°C to halt replication. 
For stationary phase samples, incubation of overnight cultures was continued for an additional 8 hours, except for L. biflexa, where cultures were grown until an OD₄₂₀ of 0.6. Cultures (0.5 mL, or 50 mL for L. biflexa), were centrifuged in 1.5 mL Eppendorf tubes at room temperature for 1 minute at 13,000 rpm. The supernatant was removed, and the bacterial pellets were stored at -20°C. Genomic DNA was extracted from bacterial pellets obtained from both exponential and stationary phases. For all strains except P. translucida TAC125, the GeneMATRIX Bacterial & Yeast Genomic DNA Purification Kit (EurX) was used. DNA extraction for P. translucida was performed using the DNeasy Blood & Tissue Kit (Qiagen). The extracted genomic DNA was quantified using a Qubit Fluorometer (Life Technologies), yielding DNA concentrations ranging from 30 to 300 ng/μL.
DNA Sequencing. Sequencing libraries were prepared according to the Truseq DNA PCR-Free library preparation protocol (Illumina). Equimolar pools of libraries were sequenced using a Hiseq 2500 system (paired-end 100 bp) or Nextseq 500 system (pair-end 150 bp) (Illumina), to obtain 15-20M reads per sample.

* HiC
The different bacterial strains were grown until mid-exponential phase (OD = 0.3) in 80 mL of LB. Bacteria were then treated with formaldehyde at a final concentration of 3%. Cultures were then incubated for 30 min under shaking at RT followed by incubation at 4°C during another 30 min. Formaldehyde was quenched by adding three times the volume of Glycine 2.5 M followed by an incubation of 20 min under shaking. Pellets were recovered by a centrifugation of 10 min at 10,000 x g and at 4°C, washed in PBS and re-centrifuge using the same settings. Pellets were then frozen in dry ice and stored at -80°C until use. Hi-C libraries were generated using the ARIMA kit (Arima Genome-Wide Hi-C Kit). Samples were first resuspended in 1 mL of sterile water and transferred to 2 mL precellys tubes containing glass beads of 0.1- and 0.5-mm diameter (Precellys – Bertin Technology). Cells were disrupted using the precellys apparatus (Precellys Evolution) and the following program (7500 rpm, 6 cycles 30 sec ON / 30 sec OFF, 4°C). Tubes were then centrifuged for 1 min at 1000 g and 700 µL of lysate was recovered and transferred to a new 1.5 mL eppendorf tube. Tube was centrifuged for 20 min at 16,000 g, 4°C. Supernatant was carefully removed and pellet was resuspended in 45 µL of water. We then followed the ARIMA protocol and the final library was purified using AMPure beads and eluted in 130 µL of water before processing for sequencing. HiC genomic libraries were first sheared at a mean size of 500 bp using a covaris S220 apparatus, processed on streptavidin beads using the colibri kit (ThermoFisher) as previously described 48,49. Libraries were sequenced on NextSeq apparatus (2 x 35 bp).