1. Development of EcoWI-seq as a one-step enzymatic approach for genome-wide base resolution PT modification mapping

In a previous work [21], we characterized the phosphorothioate-dependent restriction endonuclease (PTDR) EcoWI which cleaves at a fixed distance from its recognition sequence GpsAAC/GpsTTC. The enzyme shows specific and consistent activity on PT-modified GpsAAC/GpsTTC motifs in synthesized oligonucleotides and plasmids. Based on these findings, we developed a high throughput sequencing method named EcoWI-seq to identify genome-wide instances of PT modification at GAAC/GTTC motif. The general concept of the method is illustrated in Fig 1. Briefly, the one-step EcoWI treatment introduces a nicking site seven bases downstream of the PT GpsTTC motif and a nicking site 6 bases upstream of a PT GpsAAC motif thus creating a double-strand break with a single nucleotide 3’ end overhang. This digestion results in the pile-ups of fragments ending/starting specifically at the enzyme-induced double-strand break. It has been previously reported that digestion with EcoWI of PT modified organisms results in a broad range of fragment sizes including very large fragments [21]. Thus, to ensure uniform coverage of the genome and a quantifiable representation of all the cut sites irrespective of the cutting frequencies, we subsequently subjected the digested DNA to random fragmentation by sonication. After standard Illumina library preparation and sequencing, the digested sites are distinguished from random fragmentation patterns using a newly developed analytical procedure.

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Fig 1. Schematic illustration of EcoWI-seq.

The figure showed an example of a 5’ GpsTTC site (complement GpsAAC). Solid stars represented the PT modifications. EcoWI digestion introduced a double-strand break resulting from two opposite cut sites (indicated by arrows) located at N7/N6 downstream of the PT modified site. Additional cut(s) may occur upstream of a PT GpsTTC motif and/or downstream of a PT GpsAAC motif resulting in either an additional nick or cut respectively [21]. In the former case, the nick will be repaired during the end-prep step during library preparation and will not interfere with the double-strand cleavage pattern (see discussion). The digested gDNA is further sonicated into fragments to obtain appropriately-sized fragments before adaptor ligation. The resulting library is then amplified and sequenced using Illumina platform. Dashed stars showed amplification of fragments generated by EcoWI cuts. The EcoWI cleavage sites resulted in pile-ups of reads start/end that can be differentiated from random breaks generated from sonication.

https://doi.org/10.1371/journal.pgen.1010389.g001

2. Identification of PT modified positions by EcoWI-seq at single base resolution in the Salmonella enterica serovar Cerro 87 (SenC87) genome

We applied EcoWI-seq to analyze PT modifications in the wild type (WT) Salmonella enterica serovar Cerro 87 (SenC87) genome (Dnd+). This genome has been previously demonstrated to have PT modification at GAAC/GTTC motif (between G and A or T) [3] and a number of studies have characterized the PT modification positions in the genome using SMRT sequencing and/or iodine-induced sequencing method [16,17]. Additionally, a control experiment was performed on the same starting material using the EcoWI-seq protocol with the omission of the EcoWI enzyme (no-enzyme control). To evaluate the enzyme specificity for PT modification, we also performed EcoWI-seq experiment paired with a no-enzyme control on a dnd mutant SenC87 strain for which the dndE gene has been deleted (ΔdndE) and thus bears no PT modification in its genome (Dnd-). We performed EcoWI-seq on 1 μg of genomic DNA extracted from a 50 ml culture grown on LB medium to stationary phase. Sequencing of all libraries was performed on Illumina instruments and resulted in ~ 10 million paired-end reads per library. Reads were mapped to the SenC87 reference genome and trimmed to leave the most 5’ end mapped base.

We showed that nucleotide bias upstream and downstream of positions with high read pile-ups (>50 5’ ends) match the GAAC and GTTC motif in the genome of the Dnd+ WT strain, which is consistent with the reported enzyme specificity (Figs 2A and S1A). In contrast, no read pile-ups were observed in the no enzyme control, indicating that the pile-ups of 5’ end of reads at specific positions are resulting from the enzyme activity (S1A Fig). We confirmed that read pile-ups remained at low base levels in the genome of the ΔdndE mutant strain (S1A Fig), indicating that PT modifications are necessary for the enzyme activity. These results together demonstrate that EcoWI selectively recognizes and cuts PT modified GpsAAC/GpsTTC sites in the genome. These results also indicate that the distinctive 5’ end pile-ups at specific sites, in spite of random distribution of reads generated from sonication, can be used to identify PT modification.

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Fig 2. Genomic mapping of PT modification at single base resolution by EcoWI-seq.

(A) Motif logo of positions with more than 50 5’ end reads in the genome of WT Dnd+ SenC87 strain. (B) Plots of modification scores in the genome of WT Dnd+ (top) and ΔdndE mutant (bottom) strains. Dashed line showed the modification score cut-off used to call PT-modified sites. (C) Numbers of identified PT-modified sites by category.

https://doi.org/10.1371/journal.pgen.1010389.g002

Towards this end, we devised a modification score for every position p, which is defined as the relative ratio between the number of 5’ end reads at a position p and the median number of 5’ end reads within a +/- 50 bp window from the position p. We complemented this modification score with a strategy for the EcoWI-seq signal deconvolution step to call PT modified positions in genomes (S1B Fig). This calculation allows for the identification of PT modification at single base resolution. In agreements with previous results [6,17,18] the profile of modification scores in the Dnd+ WT SenC87 genome demonstrated that the majority of GAAC/GTTC motifs were either unmodified or modified at a very low level (modification score <5) (Figs 2B and S2A). In total, we identified 3973 predicted PT-modified sites in the WT and only one predicted modified site in the genome of the ΔdndE mutant strain (Figs 2C and S2B, and S1 and S2 Tables). We confirmed the reproducibility of the method in the Dnd+ WT SenC87 strain with independent replicates showing a good correlation (Pearson’s coefficient = 0.95, S2C Fig). Furthermore, PT-modified positions identified in the two replicates displayed 97% overlap.

Among the PT-modified sites in the genome of the Dnd+ WT SenC87 strain, 97.4% (3870) were located at the GAAC/GTTC motif, indicating the precision of EcoWI in recognizing PT modified consensus sequence (Fig 2C). The PT-modified GAAC/GTTC sites account for 12% of all possible GAAC/GTTC sites (32795 in total), which is also in accordance with previous observations [16,17].

To understand the remaining 2.6% of modified sites located at positions other than the canonical GAAC/GTTC motif, motif logo analysis was performed on these non-canonical positions and a GTAC motif was identified (S2D Fig). Interestingly, a previous study also showed that GpsTAC accounted for 6% of SenC87 PT modified sites, which was the third highest motif after the canonical GpsAAC and GpsTTC motifs [6]. It had been previously reported that some restriction enzymes (e.g. BamHI) could cleave some related sites by star activity [26]. To investigate if this is the case of EcoWI, we designed oligonucleotides containing modified GpsTAC motif or unmodified GTAC motif as control and performed EcoWI cleavage assay (S2E Fig). The GpsTAC oligonucleotide is partially cut while the unmodified GTAC oligonucleotide is not digested, demonstrating that EcoWI has activity on modified GpsTAC but not on unmodified GTAC motif at high enzyme concentration.

3. Validation of EcoWI-seq identified PT modification positions with SMRT sequencing

To validate the EcoWI-seq method, SMRT sequencing was performed on the same WT Dnd+ SenC87 strain gDNA sample used for EcoWI-seq. We recorded the interpulse durations (IPDs) of the polymerase kinetics and followed the analytical method that has previously been published for detecting PT modification in SMRT sequencing data [16] (S3A Fig and S3 Table). As expected, we observed high kinetic signals consistently occurring at GAAC/GTTC context and obtained 3473 GAAC/GTTC sites called as modified. When comparing the specific modified positions called by EcoWI-seq and SMRT sequencing, we found 1) that the total number of modified sites identified by the two methods are very similar (3473 compared to 3973) (Fig 3); 2) a very high agreement at base resolution between the two detection methods (98.1% of SMRT modified positions are also detected in EcoWI-seq, p value = 10^^-9197.425, hypergeometric test) (Fig 3); and 3) that the distributions of PT modified positions identified by the two methods are found across the genome (S3B Fig). We also searched for residual EcoWI-seq signals on SMRT unmodified positions and, in agreement with the absence of modification at these positions, found no increase of read piled-up in EcoWI-seq (S3C Fig).

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Fig 3. Validation of EcoWI-seq called PT modification positions in the WT Dnd+ SenC87 strain.

Venn plot showing the overlap of modified GpsAAC/GpsTTC positions identified using EcoWI-seq (red) and SMRT sequencing (blue). The numbers of called modified sites and overlaps were listed in the table below. The percentages of SMRT-sequencing sites overlapping with EcoWI-seq sites are listed in parentheses. Note that we also randomly chose 3473 positions from available GAAC/GTTC positions and this only led to a small percentage of overlap with EcoWI-seq modified positions.

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We confirmed that 40% (30 out of 76, p value = 4.13e-18, hypergeometric test) of the GTAC sites identified as modified in EcoWI-seq also possessed high kinetic signals in SMRT sequencing. Therefore, it is possible that this small fraction of non-canonical GAAC/GTTC “modified” sites by EcoWI-seq was attributed to the complex effect of relaxed recognition of EcoWI at preferably modified GpsTAC sequences. However, the accuracy of EcoWI-seq was not undermined as shown in the S3D Fig.

Together, these data validated the sensitivity and accuracy of EcoWI-seq in detection of PT modifications with another standard method–SMRT sequencing.

4. Optimization and characterization of EcoWI-seq

In efforts to optimize the EcoWI-seq performance, we constructed EcoWI-seq libraries using gDNA isolated from WT Dnd+ SenC87 strain and adjusted incubation time and DNA amounts. We also analyzed data with distinct sequencing coverages and sequencing methods (single end vs paired end). As shown in S4A Fig, the number of modified sites detected by EcoWI-seq increased with prolonged enzyme treatment. The majority of modified sites can be detected with 3 hours incubation and demonstrated over 93% consistency with SMRT sequencing called modification sites (S4A and S4B Fig). Testing of library constructions suggested that EcoWI-seq was able to achieve consistent and most modification sites with a minimized 500 ng DNA input (S4C Fig). In order to get optimal performance, a minimal coverage of 200x was required in the sequencing (S4D Fig). We also investigated whether single-end vs paired-end sequencing affect the identification of PT modified sites. Our results showed that the total number of predicted modified sites and the accuracy of the predictions (validated by SMRT sequencing) are similar for single-end and paired-end sequencing. These data demonstrated the consistent performance of EcoWI-seq analysis regardless of sequencing method, therefore allowing flexibility in the selection of sequencing options (S4E Fig).

5. EcoWI-seq can be applied to de-novo assembled genome

To explore whether EcoWI-seq analysis can be applied to genomic DNA for which no reference genome is available, we directly assembled the EcoWI-seq sequencing reads from experiments done on WT Dnd+ SenC87 strain using the de novo assembler SPAdes [27]. We first confirmed the quality of the assembly which demonstrated a span of nearly the size of the actual genome (S5A Fig). Next, reads were mapped back to the de novo assembled genome and PT modification positions were identified. Locations of modified positions found in the genome assembly show a 97.6% concordance compared to using the reference genome (S5B Fig). Therefore, EcoWI-seq can be used with similar accuracy on bacteria for which a reference genome is unavailable.

6. PT modification occur at consistent locations while maintaining balance

It is known from this and previous work, that only a small fraction of GAAC/GTTC motifs are PT modified. While the plasticity of PT modification in epigenomes in certain strains of bacteria has been investigated [6,7], it is still unclear how PT-modifying Dnd proteins select the targets and whether or not the establishment of modified positions is a stochastic process that is subsequently maintained. To address this question, we use a previously established in vivo PT modifying system for which the expression of the SenC87 dnd gene clusters in E. coli leads to a dnd+ strain [21]. To ensure the independent establishment of PT modifications, we conducted three independent transformation experiments respectively with SenC87 dnd or pBC4 (pUC19 with an adenovirus DNA insert). We also included two technical replicates for each SenC87 dnd transformed strain to rule out possible variations due to technical reasons (Fig 4A).

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Fig 4. Investigation of target modification sites in E. coli heterologous PT modifying system.

(A) Experiment design. Stars represent PT modifications in the genome. (B) Correlations among the 6 SenC87 dnd/pBC4 transformed E. coli strains. For each pair of comparisons, modification scores of all possible GAAC/GTTC sites were used for correlation calculation. Color bar on the side indicated the Pearson correlation values. For SenC87 dnd transformed E. coli strains, correlations were also performed among the technical duplicates (rep1 and rep2). (C) Output of Two Sample Logo [31]: Graphical representation showing statistically significant (p value < 0.01) differences in position-specific symbol compositions between modified and unmodified GAAC positions (top) and modified and unmodified GTTC positions (bottom) in E. coli.

https://doi.org/10.1371/journal.pgen.1010389.g004

We found that modifications were detected on a small subset of GAAC/GTTC sites in SenC87 dnd transformed E. coli gDNA (S6A and S6B Fig). This result indicates that the expression of the dnd gene in E. coli results in a genomic pattern of PT modifications similar to the SenC87 strain. As expected, no modification position was identified in cultures transformed with control plasmid pBC4 (S6A and S6B Fig).

Comparison of modification scores at GAAC/GTTC positions for the three independent transformations reveals that the modified GAAC/GTTC positions are highly correlated among the three SenC87 dnd transformed E. coli samples. Moreover, the correlation levels between independent transformations are equivalent to the correlation levels of the technical replicates indicating that the establishment of modified positions is non-random (Fig 4B).

If the establishment of modified positions is deterministic, we sought to identify the factor(s) that controls which positions are modified starting with possible sequence context additional to the GAAC/GTTC motifs. Towards this end, we analyzed the sequence variability (+/- 20 bp) between PT modified and unmodified GAAC/GTTC motifs in SenC87 dnd transformed E. coli strains and observed nucleotide preferences immediately upstream and downstream of the modified GAAC/GTTC motif with C and G respectively are over-represented in the modified motifs at -2 and +2 positions relative to the modified compared to unmodified GAAC/GTTC motifs. The -1 bp and +1 bp positions at modified GAAC/GTTC motifs have preference for M (A or C) and K (T or G) respectively (Fig 4C). Similar sequence preferences also applied to the PT modified positions in the genome of the SenC87 WT strain, suggesting a common mechanism in selection of modification sites despite the different targeting genomes (S6C Fig).

Bacteria strains, plasmids and Enzymes

The wild-type Salmonella enterica Cerro 87 strain was kindly provided by Dr. Toshiyki Murase. The gDNA of ΔdndE mutant Salmonella enterica Cerro 87 was kindly provided by Dr. Xinyi He (mutant gDNA, Shanghai Jiaotong University). The mutant gDNA was purified by a FastPure bacteria DNA isolation kit (Vazyme #DC103). The dnd gene clusters responsible for E. coli PT modification were cloned from Salmonella enterica Cerro 87 into a pLacZZ (modified from pUC19) backbone and validated as described previously [21]. pLacZZ-dnd+ and the control plasmid pBC4 (pUC19 with an adenovirus DNA insert) were transformed into E. coli T7 Express competent cells (Dcm-, NEB #C2566) and plated on agar plates with ampicillin selection (100 μg/ml). Plasmid transformation was carried out according to the manufacturer’s protocol (NEB). The purification of PT-dependent restriction enzyme EcoWI was performed as previously described [21].

Genomic DNA extraction and plasmid isolation

For SenC87 gDNA extraction, 50 mL cell culture were grown in LB to stationary phase and processed with the NEB Monarch genomic DNA purification kit (NEB #T3010). For genomic DNA extraction of E. coli cells, 1 ml overnight culture of pLacZZ-dnd+ or pBC4 transformed E. coli C2566 cells was harvested by centrifuging at 12,000 g for 1 min. The cell pellet was resuspended in 10 μl lysozyme (25 mg/ml) and 190 μl TE buffer, following incubation at 37°C for 5 min. Then the lysis buffer containing 265 μl TE, 15 μl 20% SDS and 3 μl 20 mg/ml Proteinase K (NEB #P8107) was added to the sample and the mixture was incubated at 37°C for 30 min with agitation at 1400 rpm to completely lyze the cells. An additional 2 μl RNase A (20 mg/ml, NEB #T3018) was added and followed by 5 min incubation at 56°C to remove residual RNA in the sample. Genomic DNA was extracted with 2 rounds of equal volume phenol/chloroform extraction, one round of equal volume chloroform, precipitated in 95% ethanol and washed with 70% ethanol. The genomic DNA was dissolved in TE buffer (pH 8). For plasmid isolation, 1–3 ml of overnight culture was pelleted, and plasmid DNA was isolated using Monarch plasmid miniprep kit (NEB #T1010).

Library preparation for EcoWI-seq

One μg genomic DNA was used for each reaction. The pLacZZ-dnd+ plasmid was spiked in as positive control. Lambda gDNA spike-in were added to serve as negative control. The genomic DNA was first digested by 1 μl EcoWI (2.7 mg/ml) in 50 μl 1x NEBuffer 2 for 3 hours of incubation at 37°C. The digested DNA fragments were purified with oligo clean and concentrator kit (Zymo Research D4060), further sheared using 200 bp protocol with Covaris S2 Ultrasonicator in 130 μl of 0.1x TE buffer and evaporated to desired volume for illumina library preparation. One reaction of NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645) was used for 1 μg genomic DNA input. The DNA library was purified with 1x volume of NEBNext Sample Purification Beads (NEB #E7103) and eluted with 40 μl of 0.1 X TE buffer.

Illumina sequencing and reads processing

Libraries were indexed with NEBNext Multiple Oligos for Illumina (NEB #E6440) and amplified using NEBNext Ultra II Q5 Master Mix for 5 cycles. The quality and quantity of libraries were checked with Agilent TapeStation Systems with High Sensitivity D1000 reagent and screen tape (Agilent #5067–5585 and Agilent #5067–5584). The libraries were pooled for sequencing on Illumina Nextseq instrument or Illumina Novaseq instrument with paired-end reads of 75 bp or 100 bp. For each sample, we achieved 10–20 million reads.

Reads were downloaded as FASTQ files and trimmed with Trim Galore v0.6.4 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The option—paired were used when processing paired end reads. Trimmed reads were aligned to the reference genome with BOWTIE2 v2.3.5.1 (26), together with SAMTOOLS V1.9 [32] to sort and index bam files for later analysis.

Calculation of PT modification scores and calling of PT modification positions in EcoWI-seq data

To calculate the modification score per position, we first computed the read depth at each position using samtools-depth and counted the number of 5’-end reads at each position using bedtools. The read depths and 5’-end reads were split by strand. Then, the median number of 5’-end reads within a +/- 50 bp window for each position was achieved or replaced with value 1 if it resulted in 0. The modification score at position p was defined as follows: (modification score)_p = (number of 5’-end reads)_p / (median number of 5’ end reads)_p. The determination of PT modification positions in EcoWI-seq depends on two cutoffs: 1) with the reads cutoff, positions with read depths less than the reads cutoff are excluded; 2) with the modification score cutoff, we only select positions with modification scores greater than the score cutoff and their -2 bp positions must also have modification scores greater than the score cutoff. This is to avoid screening for positions generated other than double stranded breaks. Finally, minor shifts between +/- 2 bp were collapsed to the same PT modification site. In this work, we used 50 as the reads cutoff for all samples (including SenC87 and E. coli dnd+). The choice of modification score cutoff varied from different samples: 20 was used for all SenC87 WT or mutant samples and the score cutoff was set from 30–60 for E. coli dnd+ samples. The scripts and detailed documentation of the analysis are available at https://github.com/rosayi/EcoWI-seq.

Library preparation for SMRT sequencing

SMRTbell libraries were constructed from SenC87 gDNA samples sheared to 10–20 kb using the G-tubes protocol, end repaired, and ligated to PacBio hairpin blunt adapter. Incompletely formed SMRTbell templates and linear DNAs were digested with a combination of Exonuclease III and Exonuclease VII (NEB #M0206 and #M0379). DNA qualification and quantification were performed using the Qubit fluorometer (Invitrogen) and 2100 Bioanalyzer (Agilent Technology). The 10 kb SMRTbell library was prepared according to PacBio 8–10 kb sample preparation protocols including additional separation on a BluePippin (Sage Science), originally sequenced using C4-P6 chemistry (2 SMRT cells, 300-minute collection time).

Detection of PT modification in SMRT sequencing data

PacBio reads were mapped to the SenC87 reference genome (GenBank: CP008925.1) and mapped reads were analyzed using the SMRTProtal motif and modification analysis module version 2.3.0. IPDs were measured and processed as described [33,34] for all pulses aligned to each position in the reference sequence. The Pacific Biosciences’ SMRTPortal analysis platform v. 2.3.0 uses an in silico kinetic reference, and a t-test based kinetic score detection of modified base positions (details are available at http://www.pacb.com/pdf/TN_Detecting_DNA_Base_Modifications.pdf). For calling of PT modified GAAC/GTTC sites, we plotted the kinetic signals at each GAAC/GTTC site on opposite DNA strands, and applied the same strategy as previously described [16] (see S3A Fig).

Comparison of modified positions

Candidate PT-modified GAAC/GTTC positions called from EcoWI-seq and SMRT sequencing methods were mapped to SenC87 reference genome (GenBank: CP008925.1) using bedtools. Overlaps between two datasets were calculated with bedtools command line or custom python script. For comparison of E. coli heterologous PT modifying samples, Pearson’s correlation was performed for each pair of samples on all GAAC/GTTC sites (including both modified and unmodified) and the correlation heatmap was plotted with python seaborn module.

Motif logo analysis

The sequences were extracted between 20 bp upstream and 20 bp downstream of a modification site and nucleotide preferences were visualized using the weblogo web tool (https://weblogo.berkeley.edu/logo.cgi) [35]. For the visualization of statistically significant nucleotides bias around modified and unmodified GAAC/GTTC sites, we generated two datasets by extracting and aligning sequences between +/- 20 bp of modified GpsAAC/GpsTTC and unmodified GAAC/GTTC sites respectively. Two sample logos (http://thetruth.ccs.neu.edu/cgi-bin/tsl/tsl.cgi) [36] was used to determine and visualize the significant differences in nucleotide composition under a t-test with p value set to 0.01.

De novo assembly

Genome de novo assembly was performed with SPAdes v3.13.0 [27] with default options. The correspondence between modified positions identified in the de novo assembled genome and the reference SenC87 genomes was done using the liftover tool flo [37].

EcoWI digestion of PT-modified oligos (GpsTAC/GpsTAC)

58mer deoxyoligonucleotides (oligos) were purchased from IDT (Integrated DNA Technologies. USA). Below is shown the DNA sequences:

Top: 5’-CCTCGAGGTCGACGGTATCGCCCGATGpsTACCTAGTCGAATTCCTGCAGCCCGGGGGA-3’

Bottom: 5’-TCCCCCGGGCTGCAGGAATTCGACTAGGpsTACATCGGGCGATACCGTCGACCTCGAGG-3’

10 ng of the duplex DNA oligos (9 nM) containing PT-modified site (GpsTAC/GpsTAC) or unmodified site (GTAC/GTAC) were digested by 5.1 μg (76 μM) and 1.7 μg (26 μM) of EcoWI in NEB Buffer 2.1 for 2 h at 37°C in a 30 μl reaction volume. The digest mix was treated with 4 μl of Proteinase K (3.2 U, NEB) at room temperature for 20 min. Half of the digested DNA was then analyzed by PAGE on a 10% TBE-PAG (Thermo-Fisher). DNA fragments were stained by SYBR Gold (Thermo-Fisher) and imaged on a Typhoon imager (GE Health). The same duplex oligos were also digested by EcoRI-HF (RI, 40 U, recognition sequence GAATTC, NEB) as a positive digestion control for 2 h at 37°C in NEB CutSmart buffer. 5-bp DNA ladder was purchased from Thermo-Fisher.