Rapid, User-friendly, Cost-effective DNA and Library Preparation Methods for Whole-Genome Sequencing of Bacteria with Varying Cell Wall Composition and GC content Using Minimal DNA on the Illumina Platform

doi:10.21203/rs.3.rs-5159516/v1

Background

Whole-genome sequencing via high-throughput sequencing is essential for identifying and characterising chromosomes and plasmids in nosocomial and environmental bacterial pathogens. To expedite outbreaks investigations, including accidental or intentional bacterial release, without compromising sequencing quality, we evaluated a more time-efficient, user-friendly, and cost-effective approach, using minimal DNA (~ 1 ng) from a single bacterial colony. Four DNA extraction methods were compared: the automated nucleic acid extractor (EZ1 Advanced, Qiagen) with or without DNA purification using AMPure® beads (EZ1 vs. EZ1-AMP), and two rapid and inexpensive methods: heat shock (HS), and glass bead disruption (GBD). Additionally, we evaluated four library preparation kits: Illumina DNA Prep (DN), Illumina Nextera XT (XT), Roche KAPA HyperPlus (KP), and NEBNext® Ultra™ II FS DNA Library Prep Kit (NN).

Results

Whole-genome sequencing performance was evaluated on Bacillus cereus, Staphylococcus epidermidis, and Enterobacter cloacae ATCC strains. Key performance indicators included sequencing depth evenness across chromosome and plasmids (accounting for GC bias), genome assembly quality measured by contig number, N50, genome fraction, and percentage of mismatches. Key performance indicators confirmed that DNA and library preparation methods significantly influenced WGS quality. GBD enabled efficient sequencing across all the three bacterial species, while HS proved inadequate for spore-forming bacteria B. cereus. DN, KP, and NN produced high-quality results with low GC bias, whereas XT exhibited significant GC bias and lower quality for bacteria with low GC content.

Conclusions

This study highlights the importance of selecting suitable DNA and sequencing library preparation methods based on bacterial cell wall composition and GC content for optimal HTS outcomes.

ATCC bacteria

Bacillus

library preparation kits

extraction methods

whole genome sequencing

plasmid

sequencing depth.

In case of infection, accurate identification and characterisation of microorganisms is critical for providing effective patient care and ensuring adherence to appropriate biosafety procedures and guidelines. Over the past decade, whole-genome sequencing (WGS) using high-throughput sequencing (HTS) has emerged as a key tool in clinical diagnostic laboratories. It enables rapid and precise nosocomial pathogen identification and characterisation, antimicrobial resistance profiling, and epidemiological investigations [1, 2]. The technology’s high resolution and rapidly decreasing costs have expanded its applications beyond clinical microbiology, making it indispensable in public health microbiology laboratories for surveillance, outbreak investigations, and transmission tracking. Furthermore, HTS proved useful in zoonotic surveillance [3] and the food safety by detecting foodborne pathogens and reducing foodborne disease outbreaks [4].

HTS technology enables the sequencing of multiple pathogen genomes in a single run. However, the quality of sequencing data heavily depends on preparatory steps such as DNA extraction and library preparation. Traditional bacterial genome sequencing methods rely on complex kit-based DNA extraction from liquid cultures. These methods are intended to eliminate potential confounding factors, such as ethylenediaminetetraacetic acid (EDTA), that may interfere with subsequent processes. To address the need for rapid and resource-efficient processing, novel DNA preparation methods such as Heat Shock (HS) lysis [5–7] and mechanical lysis using glass beads disruption (GBD), also known as bead beating [8] have been introduced. In HTS workflows, the method used for preparing a library also has a significant impact on the quality of WGS results [9, 10]. Several library preparation kits have been developed that use enzymatic-based methods such as transposases or endonucleases for fragmentation. Each method has distinct advantages, depending on the application and sequencing objectives.

This study aims to offer valuable insights into the optimal combination of DNA preparation and library preparation methods for cost-effective and accurate bacterial chromosome and plasmid sequencing. To achieve our goal, WGS was carried out on single colonies from three representative bacterial species: Bacillus cereus, Staphylococcus epidermidis, and Enterobacter cloacae, using minimal starting DNA material (1 ng). Four DNA preparation methods were compared: DNA purification from liquid cultures using a Qiagen kit (EZ1) and assessing the impact of a super-purified DNA obtained through additional treatment with AmPure XP beads (EZ1-AMP). Additionally, we compared two rapid DNA preparation methods: Heat Shock lysis (HS) and mechanical lysis with glass beads (GBD). For each DNA preparation method, we tested four library preparation kits, including two enzymatic DNA fragmentation kits (Kapa™ HyperPlus kit (KP) and NEBNext® Ultra™ II FS DNA Library Prep Kit (NN) and two tagmentase-based kits Nextera XT DNA Library Prep Kit (XT) and Illumina DNA Prep kit (DP).

Reference bacterial species

For this study, three biosafety level 1 (BSL-1) ATCC strains of Gram-positive and Gram-negative bacteria with varying GC content and genome sizes were selected: Bacillus cereus ATCC 14579 (B. cereus) (35% GC), Staphylococcus epidermidis ATCC 12228 (S. epidermidis) (32% GC) and Enterobacter cloacae ATCC 13047 (E. cloacae) (54% GC) (Table 1).

Table 1. Key bacterial features of species used in the study.

Bacterial Strain	ATCC number	ATCC Genomic Element	ATCC size	ATCC GC content (%)	NCBI Reference Sequence	NCBI Genomic Element
B. cereus	14579	Chromosome	5 416 622	35.3	NC_004722.1	Chromosome
B. cereus	14579	Plasmid 1	15200	38.0	NC_004721	pBClin15
E. cloacae	13047	Chromosome	5 317 012	54.8	NC_014121.1	Chromosome
		Plasmid 1	199 566	52.5	NC_014107	pECL_A
		Plasmid 2	86 314	47.4	NC_014108	pECL_B
		Plasmid 3	5 128	37.0	No equivalence	No equivalence
S. epidermidis	12228	Chromosome	2 503 245	32.1	NC_004461	Chromosome
		Plasmid 1	27017	29.1	NC_005004	pSE-12228-05
		Plasmid 2	21979	28.9	NC_005005	pSE-12228-04
		Plasmid 3	8007	35.6	NC_005006	pSE-12228-03
		Plasmid 4	6585	31.5	NC_005003	pSE-12228-06
		Plasmid 5	4679	31.8	NC_005007	pSE-12228-02
		Plasmid 6	4439	30.1	NC_005008	pSE-12228-01

Bacterial cultures and DNA extraction

B. cereus and E. cloacae were grown on LB-agar medium, while S. epidermidis was cultured on TSA medium, all overnight at 37°C. Petri dishes with bacterial colonies were then kept at 4°C.

Four methods of DNA preparation or extraction were performed on single bacterial colonies. Two methods involved rapid bacterial DNA lysis performed without prior DNA extraction: heat shock lysis (HS) and glass bead disruption (GBD). Additionally, two purified DNA preparations were obtained using an automatic nucleic acid extractor (EZ1 Advanced, Qiagen, Hilden, Germany). These were processed either as a one-step process (EZ1) or as a two-step process (EZ1-AMP), which included additional purification with AMPure® beads.

Heat shock bacterial lysis (HS)

A single colony was resuspended in a tube with 100 µL of molecular grade water. The suspension was heated in a water bath at 100°C for 10 minutes and then immediately placed on ice. Subsequently, the suspension was centrifuged at 18,500 x g for 5 minutes, and the supernatant containing the lysate was collected and stored at -20°C until use.

Bacterial lysis using glass bead disruption (GBD)

Mechanical disruption with glass beads was used to extract DNA as previously described by Köser et al., 2014 [11]. Briefly, a single colony was resuspended in a tube containing a volume of 8.33 µL of glass beads (425-600 µm) and 25 µL of DNA-free water for a final bead-to-water ratio of 1:3. The sample was vortexed at speed 6 on a Vortex Multi Reax (Heidolph Instruments, Schwabach, Germany) for 5 min and subsequently centrifuged in a benchtop centrifuge for 2 min at 18,500 x g. Finally, 10 µL of the supernatant was collected and stored at -20°C until use.

Bacterial purification using automatic nucleic acid EZ1 extractor (EZ1)

From a single colony, an overnight liquid culture at 37°C was performed on LB broth for B. cereus and E. cloacae, and in TSB for S. epidermidis. The culture was then centrifuged at 1,900 x g at room temperature for 10 minutes, and the supernatant was discarded. For Gram-positive bacteria, the pellet was resuspended in a solution containing 170 µL of buffer G2 (Qiagen, Hilden, Germany), 20 µL of lysozyme 50 mg/ml (Roche Diagnostics GmbH, Mannheim, Germany) and 10 µl of lysostaphin (5 mg/ml) (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel), and incubated at 37°C for 30 min with shaking at 300 rpm. For Gram-negative bacteria, the pellet was resuspended in 200 µL of buffer G2. The lysate was then purified on the EZ1 Advanced XL apparatus (Qiagen, Hilden, Germany) using the pre-programmed DNA Bacteria Card protocol and the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The elution volume was set at 100 µL, and the eluate was stored at -20°C until use.

AMPure® bead purification (EZ1-AMP)

Fifty µL of DNA extracted according to the EZ1 protocol was purified at a ratio of 1.8:1 of AMPure® beads (Agencourt® AMPure®, Beckman Coulter, MA, USA) relative to DNA, aiming to remove fragments smaller than 100 bp. The elution volume was adjusted at 50 µL, and the eluate was stored at -20°C until use.

DNA quantification and dilution

DNA quantification was performed using the Qubit 1X dsDNA High Sensitivity Assay Kit (Invitrogen Life Sciences, Merelbeke, Belgium) on a Qubit 4 Fluorometer (Invitrogen Life Sciences, Merelbeke, Belgium), following the manufacturer’s instructions with a 2 μL sample volume. Based on the DNA concentration stocks, dilutions were made in DNA-free Tris-HCl 10 mM to adjust the working solution to 0.333 ng/μL. Four aliquots of each DNA solution were prepared for use in library preparation and stored at -20°C.

Library preparation for WGS

Four library preparation kits using different fragmentation methods were used for this study: Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) (XT), DNA Prep Kit (Illumina, San Diego, CA, USA) (DP), KAPA HyperPlus Kit (Roche Diagnostics GmbH, Mannheim, Germany) (KP), and NEBNext® Ultra™ II FS DNA Library Prep Kit (Bioké, Leiden, The Netherlands) (NN). Each library was prepared using a minimal starting quantity of DNA (1 ng), with amplification conditions adapted according to the manufacturer’s instructions (Table 2).

Table 2. Library preparation kits and fragmentation methods

Library preparation kits	Acronym	Company*	Fragmentation Methods	Amplification cycles (PCR)
Illumina DNA Prep	DP	Illumina	Tagmentation by transposome	12
Nextera XT DNA Library Prep	XT	Illumina	Tagmentation by transposome	12
KAPA HyperPlus	KP	Roche	Enzymes	14
NEBNext® Ultra™ II FS DNA Library Prep	NN	New England Biolabs	Enzymes	9

Caption: Fragmentation methods and protocol adaptations based on the manufacturer's instructions for the number of PCR cycles using 1 ng of DNA. *Details of the companies are provided in the section Material and Methods.

The library preparations and WGS sequencing were carried out four times for each DNA sample. The quantity and quality of each individual library were assessed using the Qubit 1X dsDNA High Sensitivity Assay Kit on a Qubit 4 Fluorometer and a High Sensitivity DNA Assay kit on a 2100 Expert Bioanalyzer apparatus, following the manufacturer’s instructions. Subsequently, the libraries were equimolarly pooled, and the quantity and the quality of each pool were assessed again using the Qubit and Bioanalyzer, respectively. Finally, each pool was loaded onto a MiSeq for a paired-end 2 x 300 bp sequencing run using the MiSeq reagent kit V3 (600 cycles).

Bioinformatic analysis

To evaluate the impact of different DNA preparation methods and library preparation kits on WGS results, we compared key performance indicators (KPIs) from two bioinformatics pipelines: reference-based mapping and de novo assembly. The KPIs included sequencing depth evenness across chromosomes and plasmids (accounting for GC bias), the number of contigs, N50, genome fraction, and percentage of mismatches.

Reference-based mapping:

Raw sequence reads were aligned to the ATCC reference genomes of the three target bacteria (Table 1) using Minimap2 v.2.17. The resulting SAM files were sorted and indexed using SAMtools v.1.6. Sequencing depth was calculated with GenomicAlignments v.1.34.1. The sequencing depth of plasmids was normalised using the average chromosomal sequencing depth as a baseline. To assess potential GC content bias, the genome was segmented into 200 bp fragments. For each segment, the local-to-average sequencing depth ratio was calculated and plotted against the corresponding GC content.

De novo assembly:

After down-sampling reads to 150.000/sample, filtered FASTQ files were assembled into contigs using SPAdes v.4.0.0. Contigs shorter than 1000 bp were discarded.to enhance assembly quality. The quality of the assemblies (i.e. N50, number of contigs, genome fraction compared to ATCC reference genomes, and the percentage of mismatches between the assemblies and the ATCC reference genomes) was evaluated using QUAST 5.0.2.

We used the ggplot2 R package to visualise and compare quality metrics across different DNA extraction and library preparation conditions.

Four DNA preparation methods, (EZ1, EZ1-AMP, GBD, and HS) were tested, generating the sufficient minimal genomic DNA (1 ng) required for preparing sequencing libraries for the three bacterial strains assessed in this study (Table 3). Due to the small amount of starting DNA, additional amplification cycles were needed to produce sufficient material for library construction. In line with the library preparation kit recommendations, the final library underwent 9 to 14 amplification cycles. Sufficient final library material for MiSeq sequencing was successfully generated for S. epidermidis and E. cloacae using any DNA preparation method and library preparation kit. However, for B. cereus, adequate final library material was only obtained with DNA isolated using the EZ1, EZ1-AMP, and GBD methods. The HS method did not yield sufficient final library material for B. cereus, regardless of the library preparation kit used (Table 3). Since each library preparation required a different number of PCR amplification cycles, which can introduce PCR duplicates, we calculated the percentage of duplicates generated. The rate of PCR duplicates across all four library preparations was extremely low, ranging from 0 to 0.3%.

Table 3. Outcomes of DNA and library preparations

Bacterial Species	DNA Preparation Method	DNA amount (ng)	Library Preparation Outcome from four trials
Bacterial Species	DNA Preparation Method	DNA amount (ng)	DP	KP	NN	XT
B. cereus ATCC 14579	EZ1	1	4/4	4/4	4/4	4/4
	EZ1-AMP	1	4/4	4/4	4/4	4/4
	GBD	1	4/4	4/4	4/4	4/4
	HS	1	0/4	0/4	0/4	0/4
E. cloacae ATCC 13047	EZ1	1	3/4	4/4	4/4	4/4
	EZ1-AMP	1	4/4	4/4	4/4	4/4
	GBD	1	4/4	4/4	4/4	4/4
	HS	1	2/4	4/4	4/4	4/4
S. epidermidis ATCC 12228	EZ1	1	4/4	4/4	4/4	4/4
	EZ1-AMP	1	4/4	4/4	4/4	4/4
	GBD	1	4/4	4/4	4/4	4/4
	HS	1	3/4	4/4	4/4	4/4

Caption: DNA was obtained for ATTC bacterial species using four DNA preparation methods (EZ1, EZ1-AMP, GBD, and HS). The library preparations were performed four times for each DNA purification method using four library preparation kits (DP, KP, NN, and XT). The numbers in the library preparation outcome columns represent the number of successful library preparations (out of four attempts) that yielded enough final libraries for sequencing in a single MiSeq run (minimum 1 nM of final libraries for each sample).

The quality of the sequencing data was compared across the different DNA and library preparation methods using indicators from two bioinformatics pipelines: read alignment against reference genomes, and de novo assembly.

Assessing WGS quality by aligning with a reference genome:

The generated reads from each of the three bacteria were analysed and compared to a reference genome (Table 1). The alignment analysis enabled the determination of the mean sequencing depth for both chromosomes and plasmids.

To evaluate the relative sequencing depth of plasmids, we calculated the ratio of the mean sequencing depth on plasmids to that on the chromosome (Fig. 1). This ratio is crucial for ensuring that plasmids are deeply sequenced without consuming an excessive number of sequencing reads, which could reduce chromosomal sequencing depth and coverage. The three ATCC strains in this study carried plasmids with varying numbers and GC content, which can affect sequencing quality (Table 1). E. cloacae ATCC 13047 showed comparable sequencing depth for both the pECL_A (199.6 kb, 52% GC) and pECL_B (84.6 kb, 47.4%) plasmids relative to the chromosome (Fig 1a). However, the third plasmid (5.1 kb, 37.0% GC, no NCBI reference), which had a lower GC content, exhibited reduced sequencing depth when using XT library preparation kit compared to the other three kits (Fig 1a). In contrast, for S. epidermidis ATCC 1228, the relative sequencing depth on its six plasmids was 3 to 500 times higher than that of the chromosome (Fig 1b).

No sequencing results were obtained from the plasmid typically found in B. cereus ATCC 14579 (data not shown). PCRs targeting two plasmid loci in B cereus ATCC 14579 confirmed the absence of gene amplification, supporting the hypothesis that the plasmid was lost in the strain used in this study.

The relationship between sequencing depth and GC content was assessed under all conditions (Gram-positive and Gram-negative bacteria, DNA preparation method, and library preparation kit) to identify potential GC content-associated bias. Bacterial genomes were segmented into 200 bp fragments, and the GC content of each segment was calculated. The local-to-average sequencing depth ratio showed that the GC content did not affect sequencing depth for three library preparation kits (DP, KP, and NN), regardless of the bacteria strain or the DNA preparation method (Fig. 2) (Supplementary figure S1). The XT kit was the only library preparation kit where sequencing depth was influenced by GC content, with GC-rich regions being more deeply sequenced than regions with low GC content (Fig. 2) (Supplementary figure S1).

Assessing WGS quality using de novo assembly analysis

To evaluate the ability to reconstruct a whole genome from sequencing reads, we conducted a second analysis using de novo genome assembly. The number of contigs was unaffected by the DNA preparation method when libraries were prepared with the DP, KP, and NN kits (Fig. 3a-c). However, libraries generated using the XT kit produced more contigs (Fig. 3a-c). Notably, when sufficient DNA was available for E cloacae (Fig. 3 b) and S. epidermidis (Fig 3c) prepared with the HS method, we observed a noticeable increase in the number of contigs when the DP kit was used for library preparation.

The N50 values were evaluated and found to be lower for genome assemblies from libraries prepared with the XT kit compared to those from the other library preparation kits (Fig. 4a-c). For the DP, KP and NN kits, N50 values across all bacteria were similar regardless of the DNA extraction method used, except for E. cloacae (Fig. 4b) and S. epidermidis (Fig 4c) when the HS method was followed by the tagmentase-based DP library preparation.

Assemblies of B cereus, E. cloacae, and S. epidermidis using the EZ1, EZ1-AMP, and GBD DNA preparation methods, combined with the DP, KP, and NN kits, achieved high genome fraction (> 95%) compared to the ATCC reference genomes (Fig. 5a-c). In contrast, assemblies obtained using the HS DNA preparation method and/or the XT kit exhibited lower genome fractions, ranging from 60% to 95%.

Except for the HS method, assemblies for each bacterium generated very low mismatch percentages compared to the ATCC reference genomes, with particularly high base-calling performance observed using the NN library preparation kit (Supplementary figure S2).

Our investigation aimed to determine the optimal combination of DNA preparation and library preparation for sequencing the bacterial genome and plasmid DNA when working with limiting amounts of DNA. The study focused on three bacteria with varying GC contents: B. cereus, S. epidermidis, and E. cloacae, which represent both Gram-positive and Gram-negative types.

The impact of DNA extraction method on WGS has already been studied by comparing various commercial DNA preparation kits [12]. Among the commercial DNA preparation protocols used in this study, EZ1 and EZ1-AMP using solid-phase reversible immobilisation (SPRI) beads yielded similar results, with little effect on library preparation efficacy and sequencing quality. Among the two rapid DNA preparation techniques, DNA prepared by mechanical disruption of bacteria using glass bead disruption (GBD) provided results comparable to other DNA purification methods. However, the boiling extraction method (HS) had some limitations. Although this method produced enough genomic DNA (1 ng) for preparing the library of all tested bacteria, no final library could be obtained from B. cereus DNA, regardless of the library preparation kit used.

Numerous studies have demonstrated the efficacy of boiling extraction in retrieving DNA from both Gram-positive and Gram-negative strains for quantitative PCR tests [5, 13, 14]. However, B. cereus, a food-borne pathogenic bacterium that is commonly found as a contaminant in food and dairy products, has a high tolerance to heat [15, 16]. This feature may be attributed to its cell wall composition, which is typical of spore-forming Gram-positive bacteria. It contains peptidoglycan, which provides greater rigidity than the cell walls of other Gram-positive bacteria such as S. epidermidis or the Gram-negative E. cloacae. The latter two bacteria produced better sequencing results, due to their cell walls being more easily broken down by boiling. Although this boiling extraction method is simple to use and sufficient from preparing for PCR testing, it should not be used for Gram-positive spore-forming bacteria such as B. cereus.

The WGS analysis showed that plasmid sequencing depth in S. epidermidis exceeded that of the chromosome. This well-known phenomenon, most likely caused by the plasmid’s higher copy number, does not always occur. In our study, plasmid sequencing depth in E. cloacae was equal to or slightly lower than chromosome sequencing depth, while no plasmid sequencing was found in B. cereus. There are several explanations for low plasmid sequencing depth or the absence of plasmid sequencing. In some bacteria, plasmids can exist in low copy numbers [17], restricting their sequencing depth. Although plasmids can benefit their bacterial hosts, replication and gene expression through host machinery are considered metabolic burdens [18]. Consequently, in the absence of selection for plasmid-encoded traits, cells lacking plasmids outcompete cells carrying plasmids, leading to plasmid silencing over time [19]. Plasmid loss is particularly common in Bacillus bacteria [20-22]. Despite observing variability in plasmid sequencing depth across the three bacterial species, this variability did not correlate with differences in DNA extraction methods or library preparation technique except for one E cloacae plasmid with a low GC content, which showed reduced sequencing depth when using the Nextera XT library preparation kit.

It is generally accepted that GC bias in high-throughput sequencing data complicates genome assembly, with problems becoming increasingly severe outside the 45–65% GC range. This causes low sequencing depth in GC- poor sequences, with genomic windows containing 30% GC having less sequencing depth than windows close to 50% GC content [23, 24]. Among traditional library preparation kits, XT is particularly sensitive to GC content [25]. The XT sequencing depth bias problem is especially severe across the entire genome for bacteria with low GC-content [26]. This GC bias can be attributed to the binding motif of Nextera XT tagmentase, which is dependent on G and C base content [26].

S. epidermidis and B. cereus have a mean GC content of 32.07% and 35.29%, respectively, but exhibit a heterogenous repartition of the GC content across the genome, with some regions being above 40 and other being lower than 20%. Our results confirmed that using the XT kit for sequencing produced lower-quality genome assemblies compared to other library preparation kits, such as KP and NN. Recent studies have also shown that the Collibri EZ kit, which uses enzymatic fragmentation like KP and NN, outperforms Nextera XT in terms of genome assembly quality [27]. Additionally, genome assembly from DP kit after HS-based DNA extraction yielded similar results to those obtained with the XT kit for S epidermidis and E cloacae. Although previous studies have shown that the DP kit generally provides superior performance metrics including assembly quality, compared to XT [9, 28, 29], the choice of DNA preparation remains crucial when using tagmentase-based preparation kits. Lower sequencing depth in some regions, leading to poor genome coverage, may prevent the detection of single-nucleotide polymorphisms and genomic regions with functional or phylogenetical significance. Attempts to reduce gaps or low-sequencing depth in some genomic regions by obtaining more sequence reads increase sequencing costs and may limit the effectiveness of genomic analyses, especially those involving a large number of samples [30]. In this study, we intentionally reduced the number of reads per sample to 150.000, creating a constrained sequencing scenario that simulates a minimal sequencing depth (approximately 16 to 35X). This approach allowed us to evaluate the effectiveness of genomic analyses, particularly in determining the threshold for complete coverage, defined as a genome fraction of 100%, even under conditions of potentially low sequencing depth in certain genomic regions. In this study, we showed that low-cost DNA preparations, such as GBD, combined with the DP, KP, and NN library preparation kits, produce sequencing results comparable to those obtained with more expensive kit-based DNA preparation methods (Table 4). The combination of rapid GBD DNA preparation with the NN library preparation kit yielded excellent sequencing results for S. epidermidis, E. cloacae, and B. cereus, while being the most cost-effective combination. Although not tested in our study, several reports have shown that reducing reaction volumes for library preparation and omitting certain clean-up steps can significantly lower the cost of sample preparation for sequencing [31-33]. Reducing overall sequencing costs could expand the use of HTS in applications where time-efficiency, user-friendliness, and cost-effectiveness are critical. These include nosocomial pathogen surveillance in hospitals, outbreak investigation and transmission monitoring by public health microbiology laboratories, and European security efforts, where rapid and unambiguous genomic and plasmid characterisation of Bacillus species, including B. anthracis, is critical in cases of accidental or intentional bacterial release.

Table 4: Cost per sample (€; excl. VAT)) for DNA and library preparation

DNA Purification Method	Price/extraction	Library Preparation including indexes
		Price/sample
		DP	KP	NN	XT
		39,20	46,05	29,91	40,35
EZ1	12,08	51,28	58,13	41,99	52,43
EZ1-AMP	13,22	52,42	59,27	43,13	53,57
GBD	0,11	39,31	46,16	30,02	40,46
HS	0,00	39,20	46,05	29,91	40,35

In conclusion, based on ATCC reference strains and two bioinformatics pipelines, our KPIs demonstrate that DNA and library preparation methods significantly affect the quality and efficiency of bacterial chromosome and plasmid sequencing. Rapid, GBD-based DNA preparation method is effective for library preparation and performs comparably to automated robotic extraction. However, the HS method is ineffective against spore-forming Gram-positive B. cereus and unsuitable for de novo assembly when using tagmentase-based preparation kits. Nonetheless, Both GBD and HS, as low-cost DNA preparation methods, remain viable options for resource-constrained and mobile laboratories. Notably, the DP kit and endonuclease-based methods consistently yield high-quality results with minimal GC bias, whereas the XT kit shows significant GC bias and lower quality in low-GC-content bacteria. These findings emphasise the importance of selecting appropriate preparation methods, particularly for bacteria with different cell wall compositions and GC content.

WGS

Whole-genome sequencing

HTS

High-throughput sequencing

EZ1

EZ1 Advanced Nucleic Acid Extractor (Qiagen)

AMP

AmPure® beads

HS

Heat shock

GBD

Glass bead disruption

DN

Illumina DNA Prep

XT

Illumina Nextera XT

KP

Roche KAPA HyperPlus

NN

NEBNext® Ultra™ II FS DNA Library Prep Kit

ATCC

American Type Culture Collection

PCR

Polymerase Chain Reaction

EDTA

Ethylenediaminetetraacetic acid

LB

Luria-Bertani (agar or broth)

TSA

Tryptic Soy Agar

TSB

Tryptic Soy Broth

SPRI

Solid Phase Reversible Immobilisation

bp

Base pairs

QC

Quality Control

B. cereus

Bacillus cereus ATCC 14579

S. epidermidis

Staphylococcus epidermidis ATCC 12228

E. cloacae

Enterobacter cloacae ATCC 13047

Ethics approval and consent to participate

Not Applicable

Consent for publication

Not Applicable

Availability of data and materials

We have not yet deposited the raw NGS data from this project in a public database, as we question the added value, given the abundance of publicly available sequence data for these ATCC reference strains and the large file sizes involved. However, if you believe these raw data would provide meaningful value to the scientific community, we are open to submitting them to the European Nucleotide Archive (ENA) or Sequence Read Archive (SRA).

Competing interest

No potential conflicts of interest were reported by the authors.

Funding

This study was funded by the project MOBVEC (Mobile Bio-Lab to support first response in Arbovirus outbreaks), grant 101099283 from HORIZON-EIC-2022-PATHFINDEROPEN-01, as well as the project TeamUp Holistic capability and technology evaluation and co-creation framework for upskilled first responders and enhanced CBRN-E response), grant 101121167 from HORIZON-CL3-2022-DRS-01-09, and the project eNOVATION (Extended Network of CBRN Training Centres for Innovation), grant 101168349 HORIZON-CL3-2023-DRS-01-04. The funder had no role in the study design, collection, analysis and interpretation of data, preparation of the manuscript, or the decision to submit the paper for publication.

Author Contributions

Conceptualisation, B.B., and J-L.G.; Methodology, B.B., J-F.D., J.A., and J-L.G.; Validation, B.B., J.A., and J-L.G.; Formal Analysis, J.A.; Resources, J-L.G.; Data Curation, J.A.; Writing – Original Draft Preparation, B.B., J.A., JF.D, and J-L.G.; Writing – Review & Editing, B.B., J.A., JF.D, and J-L.G..; Visualisation, B.B., J.A. and J-L.G.; Supervision, J-L.G.; Project Administration, J-L.G.; Funding Acquisition, J-L.G.

Acknowledgments

We extend our gratitude to Béatrice Sulka (Belgian Defence Laboratories Department) for setting up the heat shock and the glass-bead disruption method on plasmid-containing Bacillus species, and to Michèle Bouyer (Belgian Defence Laboratories Department) for her invaluable assistance in culturing bacterial isolates in the early phase of this study.

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No competing interests reported.

Rapid, User-friendly, Cost-effective DNA and Library Preparation Methods for Whole-Genome Sequencing of Bacteria with Varying Cell Wall Composition and GC content Using Minimal DNA on the Illumina Platform

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