Review of current diagnostic methods and advances in Helicobacter pylori diagnostics in the era of next generation sequencing

Table 1 Who to test, summary of the recommendations from the fifth Maastricht/Florence consensus report[23]

Recommendations from the fifth Maastricht/Florence consensus report
Dyspepsia	Subject to regional Helicobacter pylori prevalence, not applicable to patients with alarm symptoms or older patients
Peptic ulcers	Especially in aspirin and nonsteroidal anti-inflammatory drugs users with history of peptic ulcer
Gastritis	Especially in long-term proton-pump-inhibitor users
Gastric cancer	In individuals at increased risk of gastric cancer
MALToma	In individuals with localized early-stage MALToma
Iron deficiency anemia, idiopathic thrombocytopenic purpura, vitamin B12 deficiency

Table 2 Standard clarithromycin-based triple regimens using metronidazole or amoxicillin and associated costs

	Drug	Dose	Costs (EUR) per dose	Costs (EUR) for 7 d	Costs (EUR) for 14 d
Standard triple regimen (with metronidazole)	Clarithromycin	500 mg (twice daily)	1.1	15.4	30.8
	Metronidazole	500 mg (three times daily)	0.6	13.1	26.2
	Pantoprazole (Proton-pump inhibitor)	Standard dose (twice daily)	0.1	1.6	3.2
In total			1.8	30.1	60.2
Standard triple regimen (with amoxicillin)	Clarithromycin	500 mg (twice daily)	1.1	15.4	30.8
	Amoxicillin	1 g (twice daily)	1.4	20.2	40.4
	Pantoprazole (Proton-pump inhibitor)	Standard dose (twice daily)	0.1	1.6	3.2
In total			2.6	37.2	74.4

Table 3 Alternative antibiotic Helicobacter pylori eradication therapy using quadruple or levofloxacin-based regimens and associated costs

Regimen	Drug	Dose	Costs(EUR) per dose	Costs (EUR) for 7 d	Costs (EUR) for 14 d
Bismuth quadruple regimen	Tetracycline	500 mg (four times daily)	0.6	12	16.8
	Metronidazole	500 mg (three times daily)	0.6	18.6	26.2
	Pantoprazole (Proton-pump inhibitor)	Standard dose (twice daily)	0.1	2.2	3.0
	Bismuth Subsalicylate	Standard dose (three times daily)	0.3	10.0	14.0
In total			1.6	42.8	60.0
Levofloxacin-based regimen	Levofloxacin	500 mg (once daily)	2.7	27.0	38.0
	Amoxicillin	1 g (twice daily)	1.4	28.8	40.4
	Pantoprazole (Proton-pump inhibitor)	Standard dose (twice daily)	0.1	2.2	3.1
In total			4.2	58.0	81.5
Concomitant regimen	Clarithromycin	500 mg (twice daily)	1.1	22.0	30.8
	Amoxicillin	1 g (twice daily)	1.4	28.8	40.4
	Metronidazole	500 mg (three times daily)	0.6	2.2	3.0
	Pantoprazole (Proton-pump inhibitor)	Standard dose (twice daily)	0.1	18.6	26.0
In total			3.2	72.0	100.0

Table 4 A PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND transcriptome OR transcriptomic” yielded 12 original research studies

Study	Main finding	Method	Sequencing	Ref.
1	Objective	H. pylori 26695 culture grown in liquid medium to log phase	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Estibariz et al[107]
	Characterization of the MTase JHP1050 in H. pylori
	Main finding
			RNA-sequencing on an Illumina HiSeq platform (Illumina, United States; 1 × 50 bp)
	The MTase JHP1050, which methylates GCGC sequences, was found to be highly conserved in all analyzed H. pylori strains, with a nucleotide sequence identity > 87%. Absence of m5C methylation had a significant effect on H. pylori growth, led to a significant reduction in DNA uptake capacity, and reduced the bacterial protection against an excess of copper
2	Objective	H. pylori 26695 culture grown in liquid medium to early log phase	RNA-sequencing on a HTSeq v0.6.1 platform[148]	Han et al[114]
	Analyzing the impact of bismuth on a diverse array of intracellular pathways in H. pylori
	Main finding
	Bismuth influences multiple metabolic pathways and suppresses energy production in H. pylori through disruption of the central carbon metabolism of the bacterium. Bismuth initially perturbs the citric acid cycle and then urease activity, followed by the induction of oxidative stress and inhibition of energy production, and in the meantime, induces extensive down-regulation in the H. pylori metabolome
3	Objective	H. pylori grown on non-selective solid agar media	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina) RNA-sequencing on an Illumina NextSeq platform (Illumina)	Hathroubi et al[106]
	Transcriptomic analysis to assess the process of biofilm formation in H. pylori
	Main finding
	H. pylori biofilm cells displayed a distinct transcriptomic profile. Lower metabolism and stress responses, likely associated with the microenvironment generated in the H. pylori biofilm, could be determinants of antimicrobial tolerance and involved in the persistence and survival of H. pylori. However, there were no specific genes up-or downregulated that are specific for biofilm formation, suggesting that there is no biofilm-specific set of genes expressed. However, genes encoding flagellar filaments were upregulated in biofilm cells and formed an integral part of the biofilm matrix
4	Objective	H. pylori strain 7.13 was grown in liquid medium to mid exponential phase (OD of 0.5)	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Loh et al[111]
	Transcriptional analysis of H. pylori gene expression under high salt conditions
	Main finding
	Differential expression of multiple genes encoding outer membrane proteins, including adhesins (SabA, HopA and HopQ) and proteins involved in iron acquisition (FecA2 and FecA3) was observed. Transcript levels of sabA, hopA and hopQ were increased, whereas transcript levels of fecA2 and fecA3 were decreased under high-salt conditions. Functions associated with the up- and downregulated genes included acetone metabolism, acid survival, flagellar synthesis and iron transport
			RNA-sequencing on an Illumina HiSeq 3000 platform (Illumina; 2 × 75 bp)
5	Objective	H. pylori strain G27 grown in liquid medium, followed by adaptation of the pH (3.0, 4.5, 6.0, 7.4, 8.0)	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Marcus et al[112]
	Transcriptional analysis of H. pylori gene expression under different pH conditions
	Main finding		RNA-sequencing on an Illumina HiSeq 2500 platform (Illumina; 1 × 50 bp)
	About 250 genes were induced, and an equal number of genes were repressed at acidic pH. Genes encoding for antioxidant proteins, flagellar structural proteins, type-IV secretion system (T4SS)/Cag-pathogenicity island, FoF1-ATPase, and proteins involved in acid acclimation were highly expressed at acidic pH
6	Objective	Different H. pylori strains grown in liquid medium to mid exponential phase (OD = 0.7) with/without heat shock treatment	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Pepe et al[113]
	Characterization of the heat shock protein repressor (HspR) binding sites in H. pylori
	Main finding		RNA-sequencing on an Illumina GAIIX platform (Illumina; 1 × 85 bp)
	HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. There is high sequence conservation in the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.) among H. pylori strains. Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA
7	Objective	Gastric biopsy specimens from patients with H. pylori infection and premalignant tissue changes	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Thorelll et al[116]
	Analysis of the composition of the transcriptionally active microbial community and H. pylori gene expression in gastric biopsy specimens from patients with H. pylori infection and premalignant tissue changes
			RNA-Sequencing on an Illumina HiScanSQ platform (Illumina; 2 × 100 bp)
	Main finding
	Although H. pylori infection did not change the bacterial diversity, H. pylori abundance was positively correlated with the presence of Campylobacter, Deinococcus and Sulfurospirillum. Quantification of H. pylori gene expression found high expression of genes involved in pH regulation and nickel transport
8	Objective	H. pylori strains grown in liquid medium and treated with high nickel (500 μM Ni2+) concentrations	Depletion of ribosomal RNA (RiboZero, Epicentre, Illumina)	Vannini et al[115]
	Characterization of the Nickel dependent transcriptional regulator (NikR) in H. pylori
	Main finding		RNA-sequencing on an Illumina MiSeq platform (Illumina; 1 × 76 bp)
	NikR not only regulates metal-ion transporters but also virulence factors, non-coding RNAs, as well as toxin-antitoxin systems in response to nickel stimulation
9	Objective	H. pylori strains grown to mid exponential growth phase (OD = 0.7)	RNA-sequencing on an Illumina HiSeq 2000 platform (Illumina; 1 × 97 bp)	Bischler et al[108]
	Characterization of Nudix hydrolases in H. pylori
	Main finding
	H. pylori encodes two proteins resembling Nudix enzymes. One of them, HpRppH, is an RNA pyro-phosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. Transcriptional analysis revealed at least 63 potential HpRppH targets in H. pylori
10	Objective	H. pylori strains grown in liquid medium to mid exponential phase (OD of 0.5)	Depletion of ribosomal RNA by a rRNA modified capture hybridization approach from MICROBExpress kit (Ambion, Invitrogen, Life Technologies)	Redko et al[110]
	Characterization of the exo- and endoribonuclease RNase J in H. pylori and its putative targets
	Main finding
	Strong depletion of RNase J led to a massive increase in the steady-state levels of non-rRNAs. mRNAs and RNAs antisense to open reading frames. In contrast, non-coding RNAs expressed in the intergenic regions were much less affected by RNase J depletion. This suggests that RNase J is a major RNase involved in degradation of most cellular RNAs in H. pylori
			RNA-sequencing on an Illumina HiSeq 2000 platform (Illumina; 1 × 50 bp)
11	Objective Analysis of methylated DNA sites throughout the H. pylori genome for several closely related H. pylori strains	H. pylori strains (HPYF1 and HPYF2) grown to mid exponential phase (OD of 0.4)	RNA-sequencing on an Illumina HiSeq 2000 platform (Illumina)	Futura et al[149]
	Main finding
	Overall, the methylome was highly variable among closely related H. pylori strains. DNA sequence motifs for methylation could be assigned to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. Knocking out one of the Type I specificity genes led to transcriptome changes
12	Objective	H. pylori grown on non-selective solid agar media	Total RNA was digested with DNase I	Sharma et al[150]
	Characterization of the transcriptome of H. pylori, and construction of a genome-wide map of H. pylori transcriptional start sites and operons
			RNA-sequencing was performed on a Roche 454 FLX platform (Roche, Basel, Switzerland) and on a Genome Analyzer II platform (Illumina; 1 × 76 bp)
	Main finding
	Discovery of hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. An unexpected number of approximately 60 small RNAs including the epsilon-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs were discovered

Their objective, employed sequencing method and main finding is briefly described in the table. H. pylori: Helicobacter pylori.

Table 5 A PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND next generation sequencing” yielded 19 original research studies

Study	Main finding	Method	Sequencing	Ref.
1	Objective	DNA extraction from gastric biopsy specimens	Targeted 16S rRNA sequencing on an Ion S5XLplatform (Thermo Fisher Scientific, United States)	Han et al[117]
	Assessment of the correlation between the microbial gut community composition and the degree of inflammatory cell infiltration, endoscopic findings and the gastrointestinal disorders symptom severity index (PAGI-SYM)
	Main finding
	Histological and endoscopic gastritis was associated with the abundance of H. pylori and that of commensal bacteria in the stomach. The abundances of Variovorax paradoxus and Porphyromonas gingivalis were correlated with histological gastritis, but not with endoscopic or symptomatic gastritis. The total PAGI-SYM score showed a stronger correlation with the duodenal microbiota (Prevotella nanceiensis and Alloprevotella rava) than with the gastric microbiota (H. pylori, Neisseria elongate, and Corynebacterium segmentosum)
2	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina, United States)	Miftahussurur et al[128]
	Resistance to metronidazole rifaximin, rifabutin, furazolidone, garenoxacin and sitafloxacin was investigated in Indonesian H. pylori strains
	Main finding
	Furazolidone-, rifabutin-, and sitafloxacin-based therapies might be considered as alternative regimens to eradicate metronidazole and clarithromycin resistant H. pylori in Indonesian patients. Moreover, sitafloxacin but not garenoxacin should be considered for eradication of levofloxacin-resistant H. pylori strains
3	Objective	DNA extraction from gastric FFPE tissue blocks	16S rRNA targeted sequencing on an Ion Torrent (Thermo Fischer) platform	Nezami et al[132]
	Detection of H. pylori mutations that are known to confer resistance to clarithromycin, levofloxacin, and tetracycline directly from formalin-fixed paraffin-embedded (FFPE) gastric biopsy specimens using next generation sequencing
	Main finding
	Therapy failure correlated with the number of mutated genes: no failure in cases with no mutations (0/15), 19% (5/27) failure in cases with one gene mutation, and 69% (11/16) failure in cases with more than one mutated gene. Common 23S rRNA mutations (A2146G or A241G) were present in 88% (14/16) of failed cases as opposed to only 10% (4/42) of eradicated cases (P < 0.001). NGS can be used on clinical specimens collected during standard of care testing to detect mutations that correlate with increased risk of treatment failure
4	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina HiSeq 2500 platform (Illumina; 2 × 250 bp)	Yu et al[118]
	Assessment of the changes in the microbial esophagal community composition in Chinese patients with reflux esophagitis and healthy volunteers using metagenomic high-throughput DNA sequencing
	Main finding
	Moderate changes in the microbial community composition were found in patients with reflux esophagitis and compared with the healthy volunteers. At the phylum level, only Bacteroidetes differed between the groups, being less abundant in the reflux esophagitis group. The overall number and diversity of species tended to be lower in reflux esophagitis patients, but there were no significant differences between the groups. Three genera, Prevotella, Helicobacter and Moraxella, were obviously depleted in reflux esophagitis patients
5	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina)	Zhao et al[119]
	Characterization of H. pylori-induced alterations in the gastric and tongue coating microbiota and evaluation of potential impacts on human health in patients with chronic gastritis
	Main finding
	Significant alterations of the gastric microbiota were found in H. pylori-positive (cagA-positive) samples represented by a decrease in bacterial diversity, a reduced abundance of Roseburia and increased abundances of Helicobacter and Haemophilus. At the community level, functions involved in biofilm formation, mobile element content, and facultative anaerobiosis were significantly decreased in the microbial community in H. pylori-positive subjects. Presence of CagA was linked to an increased proportion of Gram-negative bacteria in the stomach, thereby contributing to an up regulation of lipopolysaccharide biosynthesis
6	Objective	DNA extraction from gastric biopsy specimens or surgical specimens of non-neoplastic gastric mucosa adjacent to the tumor	16S rRNA targeted sequencing on an Ion PGM Torrent platform (Thermo Fischer Scientific)	Ferreira et al[138]
	Characterization of the microbial community in patients suffering from gastritis and gastric cancer
	Main finding
	The gastric carcinoma microbiota was characterized by reduced microbial diversity, decreased abundance of H. pylori and the enrichment of other bacterial genera, mostly represented by intestinal commensals. The combination of these taxa into a microbial dysbiosis index revealed that dysbiosis can be used to discriminate between gastritis and gastric carcinoma. Analysis of the functional features of the microbiota was compatible with the presence of a nitrosating microbial community in carcinoma
7	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina)	Gotoda et al[142]
	Assessment of the changes in the gut microbiome after H. pylori eradication therapy in teenagers
	Main finding
	Alpha diversity revealed that both species richness and evenness were recovered to pre-treatment levels at 2 mo after H. pylori eradication therapy. Although H. pylori eradication therapy caused short-term dysbiosis, microbial diversity was restored in healthy teenagers
8	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 2 × 300bp)	Iino et al[120]
	Assessment of the association between H. pylori infection and the abundance of Lactobacillus species in the gut microbial community in Japanese patients
	Main finding
	The relative abundance of Lactobacillus in H. pylori-infected subjects with severe atrophic gastritis was higher compared with patients with mild atrophic gastritis and without atrophic gastritis (P < 0.001) and non-infected subjects (P < 0.001). The proportion of Lactobacillus salivarius was high in H. pylori-infected subjects while that of Lactobacillus acidophilus was high in non-infected subjects
9	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Aftab et al[103]
	Determination of the sequences of virulence genes (cagA and vacA) and seven housekeeping genes by next generation sequencing
	Main finding
	All H. pylori strains were considered Western-type, and 73.2% of them carried cagA. Patients infected with cagA-positive strains had more severe histological scores than patients infected with cagA-negative strains. Thus, the low incidence of gastric cancer in Bangladesh might be attributable to the high proportion of less-virulent H. pylori genotypes
10	Objective	DNA extraction from gastric biopsy specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 1 × 500 bp)	Klymiuk et al[139]
	Assessment of the bacterial microbiome in a total of 30 homogenized and frozen gastric biopsy samples from eight geographic locations.
	Main finding
	H. pylori infection of the gastric habitat dominates the gastric microbiota in most patients and is associated with a significant decrease of the microbial alpha diversity. Moreover, some bacterial genera like Actinomyces, Granulicatella, Veillonella, Fusobacterium, Neisseria, Helicobacter, Streptococcus, and Prevotella were associated with the presence of H. pylori
11	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[124]
	Characterization of levofloxacin, metronidazole, clarithromycin, amoxicillin and tetracycline resistance in H. pylori isolated from 158 dyspeptic patients in Santo Domingo
	Main finding
	Clarithromycin and amoxicillin resistance were low (3.1% and 1.6%), and no resistance to tetracycline was found. In contrast, metronidazole and levofloxacin resistance were high (82.8% and 35.9%). Most levofloxacin-resistant H. pylori strains had an amino acid substitution at codon 87 or 91 in the gyrA gene. Many different rdxA and frxA mutations in metronidazole-resistant H. pylori strains were found without synergistic effects. Novel mutations in dppA, dppB, fdxA and fdxB, irrespective of rdxA and frxA mutations were associated with different levels of metronidazole resistance in H. pylori
12	Objective	DNA extraction from stool specimens	16S rRNA targeted sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Yanagi et al[121]
	Assessment of the influence of antimicrobials on both, the gut microbiota community composition and the plasma ghrelin level in H. pylori-infected patients, who underwent eradication therapy (amoxicillin, clarithromycin and proton-pump inhibitors)
	Main finding
	The Bacteroidetes:Firmicutes (B:F) ratio was significantly increased 3 months after than before antibiotic treatment (P < 0.01). A significant decrease in the concentration of active ghrelin (P < 0.01) in the plasma was observed before and 3 mo after antibiotic therapy
13	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiSeq 2000 and MiSeq platform (Illumina; 2 × 150 bp and 2 × 300 bp)	Hashinaga et al[100]
	Comparison of cagA and vacA sequences of H. pylori strains isolated from patients with gastric cancer and MALT lymphoma in Japan
	Main finding
	Conventional genotyping of cagA and vacA showed no significant difference between patients with gastric cancer and MALT lymphoma. When comparing full protein sequences of CagA and VacA, four novel loci were found on CagA, and three loci were detected on VacA. Significant differences were observed at one CagA locus between gastritis and MALT lymphoma H. pylori strains, and at one VacA locus between gastritis and gastric cancer H. pylori strains
14	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[126]
	Characterization of levofloxacin, metronidazole, clarithromycin, amoxicillin and tetracycline resistance in H. pylori isolates from Indonesia
	Main finding
	Clarithromycin, amoxicillin and tetracycline resistance were low (9.1%, 5.2% and 2.6%). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were found. Metronidazole resistant H. pylori strains showed different rdxA amino acid substitutions, and the 23S rRNA A2147G mutation occurred in clarithromycin resistant H. pylori. However, one clarithromycin resistant H. pylori strain had a novel mutation in rpl22 without an A2147G mutation. Amino acid exchanges at N87 and/or D91 of gyrA were associated with levofloxacin-resistance
15	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina)	Miftahussurur et al[127]
	Characterization of H. pylori strains isolated from 146 patients in Kathmandu, Nepal
	Main finding
	H. pylori was not resistant to amoxicillin and tetracycline. In contrast, metronidazole resistance was extremely high (88.1%), while clarithromycin resistance was modestly high (21.4%). Most of metronidazole resistant H. pylori strains had diverse rdxA and frxA mutations. Novel mutations in dppA (A212, Q382 and I485) and dapF (L145, T168, E117, V121, R221) aside from missense mutations in rdxA were found in metronidazole resistant H. pylori strains. Amino acid exchanges at N87 and/or D91 in gyrA were predominantly found in levofloxacin-resistant H. pylori strains
16	Objective	A metronidazole-resistant strain was cultured from the metronidazole-susceptible H. pylori reference strain 26695 by exposure to low concentrations of metronidazole	Sequencing on an Illumina HiSeq 2000 platform (Illumina; 2 × 90 bp)	Binh et al[129]
	Characterization of wildtype and metronidazole resistant H. pylori reference strain 26695 in order to elucidate the molecular basis of metronidazole resistance and the involved genes in H. pylori
	Main finding
	Mutated sequences in rdxA were successfully transformed into the H. pylori reference strain 26695, and the transformants showed resistance to metronidazole. Transformed H. pylori isolates containing a single mutation in rdxA showed a low MIC (16 mg/L), while those containing mutations in both rdxA and frxA showed a higher MIC (48 mg/L). Moreover, mutations in rpsU may play a role in metronidazole resistance
17	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina platform (Illumina)	Furuta et al[104]
	H. pylori strains were isolated from the members of five families to investigate the microevolution and adaptation of the H. pylori genome using next generation sequencing and multi-locus sequence typing
	Main finding
	Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous mutations were found in virulence-related genes (cag, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes
18	Objective	H. pylori strain UM032 was grown on non-selective agar medium	Sequencing on a RS instrument (Pacific Biosciences, United States; yielding > 300 × average genome coverage) and on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Lee et al[109]
	Elucidating the biological significance of the restriction-modification (R-M) system in the physiology and pathogenesis of H. pylori
	Main finding
	Strain UM032 contains a relatively large number of R-M systems, including some MTase activities with novel specificities. Specifically, 17 methylated sequence motifs corresponding to 1 Type I and 16 Type II R-M systems were found
19	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiSeq 2000 platform (Illumina; 2 × 90bp)	Miftahussurur et al[122]
	Assessment of the prevalence of H. pylori infection and evaluation of human migration patterns in the remote areas of North Sulawesi using next-generation sequencing and multi-locus sequence typing
	Main finding
	H. pylori prevalence was low (14.3% in adults and 3.8% in children). One H. pylori strain carried East Asian type cagA (ABD type), vacA s1c-m1b, iceA1, jhp0562-positive/β-(1,3), oipA “status-on”. Phylogenetic analyses showed that the strain belonged to hspMaori type, a major type observed in native Taiwanese and Maori tribes

Their objective, employed sequencing method and main finding is briefly described in the table. H. pylori: Helicobacter pylori.

Table 6 A PubMed, MEDLINE and EMBASE search using the terms “Helicobacter pylori AND whole genome sequencing” yielded 15 original research studies

Study	Main finding	Method	Sequencing	Ref.
1	Objective	DNA extraction from biopsy specimens	Sequencing on an Illumina HiSeq 2500 platform (Illumina, United States; 2 × 50 bp)	Ailloud et al[123]
	Investigation of H. pylori evolution during infection and population dynamics inside the gastric environment
	Main finding
	Phylogenetic analyses suggested location-specific evolution and bacterial migration between gastric regions. Migration was significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggested that chemotaxis, regulatory functions and outer membrane proteins contribute to the specific adaptation to the antral and oxyntic mucosa
2	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Lauener et al[125]
	Single nucleotide polymorphisms (SNPs) were detected in H. pylori isolates by whole genome sequencing and their correlation with phenotypic resistance to clarithromycin, metronidazole, tetracycline, levofloxacin and rifampicin was assessed
	Main finding
	Overall, there was high congruence of > 99% between phenotypic drug susceptibility testing results for clarithromycin, levofloxacin, and rifampicin and SNPs identified in the 23S rRNA, gyrA and rpoB genes. However, it was not possible to infer a resistance phenotype for metronidazole based on the occurrence of distinct SNPs in rdxA and/or frxA
3	Objective	H. pylori strains grown on solid non-selective agar media	Sequencing on an Illumina HiSeq platform (Illumina; 2 × 150 bp)	Chen et al[133]
	Characterization of polymorphisms in Clarithromycin resistant and susceptible H. pylori strains using whole genome sequencing
	Main finding
	No mutations known to be associated with clarithromycin resistance, except for the controversial T2182C mutation, were detected. Single nucleotide variants (SNVs) in multidrug efflux transporter genes and HP0605 were significantly different between clarithromycin resistant and susceptible H. pylori strains. No significant difference in SNVs of membrane proteins of the RND family or MFS (HP1181) family were found
4	Objective	H. pylori strains isolated from patients with abdominal pain, gastritis, gastric or duodenal ulcers	Sequencing on an Illumina MiSeq platform (Illumina)	Quintana-Hayashi et al[151]
	Characterization of the binding ability, adhesion modes, and growth of H. pylori strains isolated from pediatric patients with abdominal pain, gastritis, gastric or duodenal ulcers
	Main finding
	Increased adhesion capacity of pediatric peptic ulcer disease (PUD) H. pylori strains to human gastric mucins compared to the non-ulcer dyspepsia (NUD) strains both at neutral and acidic pH, regardless if the mucins were positive for Lewis b (Leb), Sialyl-Lewis × (SLex) or LacdiNAc. In addition to babA positive strains being more common among PUD associated strains, H. pylori babA positive strains bound more avidly to gastric mucins than NUD babA positive strains at acidic pH. Binding to Leb was higher among babA positive PUD H. pylori strains compared to NUD strains at neutral, but not acidic, pH. PUD derived babA-knockout mutants had attenuated binding to mucins and Leb at acidic and neutral pH, and to SLex and DNA at acidic pH
5	Objective	H. pylori strain B128 isolated from a gastric biopsy of a patient with gastric ulceration was challenged with low/high salt concentrations	Sequencing on an Illumina MiSeq platform (Illumina)	Noto et al[99]
	H. pylori was continuously cultured in vitro under low iron or high salt conditions to characterize fur genetic variation. Moreover, fur sequence variation was assessed in 339 clinical H. pylori strains
	Main finding
	Exposure to low iron or high salt selected for a specific single nucleotide polymorphism in the fur gene (FurR88H) in H. pylori. Among the isolates examined, 17% of H. pylori strains isolated from patients with premalignant lesions harbored the FurR88H variant, compared to only 6% of H. pylori strains from patients with non-atrophic gastritis. These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis
6	Objective	H. pylori strains were cultured on solid non-selective agar media	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Zhang et al[152]
	Comparison of homogenization vs enzymatic digestion protocols for DNA extraction from gastric, esophageal and colorectal biopsies and survey of the microbial content and composition using whole genome sequencing
	Main finding
	Neither method demonstrated preferential extraction of any particular clade of bacteria, nor significantly altered the detection of Gram-positive or Gram-negative organisms. However, although the overall microbial community composition remained very similar and the most prevalent bacteria could be detected effectively using either method, the precise community structure and microbial abundances between the two methods were different. The homogenization extraction method provided higher microbial DNA content and higher read counts from human tissue biopsy samples of the gastrointestinal tract
7	Objective	H. pylori strains isolated from gastric biopsies of patients suffering from dyspepsia	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Kumar et al[153]
	Whole genome sequencing and comparative analysis of three H. pylori strains isolated from three Arab patients
	Main finding
	The three genomes clustered along with HpEurope strains in the phylogenetic tree comprising various H. pylori lineages. The three genomes possessed a complete cag-pathogenicity island with an AB-C type EPIYA motif
8	Objective	H. pylori strains were isolated from gastric biopsy specimens of patients with chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Ogawa et al[101]
	Characterization of cagL and cagI in H. pylori isolated from patients in Southeast Asia with chronic gastritis, gastric ulcer, duodenal ulcer and gastric cancer
	Main finding
	CagL motifs were highly conserved among the H. pylori isolates. CagL E59 and I234 in the C-terminal motif were more common in H. pylori isolates from gastric cancer patients. The CagI C-terminal motif was completely conserved across all H. pylori isolates
9	Objective	H. pylori strains were isolated from gastric biopsy specimens of patients with non-ulcer dyspepsia, gastric ulcer and duodenal ulcer	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Silva et al[102]
	Characterization of the expression of virB genes, encoding parts of the type-IV secretion system (T4SS)/Cag-pathogenicity island, in H. pylori strains isolated from Western patients with different gastrointestinal malignancies
	Main finding
	The region spanning from virB2 to virB10 constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions.
10	Objective	H. pylori culture from gastric biopsy specimens	Sequencing on an Illumina HiScanSQ platform (Illumina; 2 × 100 bp)	Thorell et al[154]
	Characterization of H. pylori strains isolated in Nicaragua
	Main finding
	The Nicaraguan isolates showed a phylogenetic relationship with West African H. pylori isolates in whole genome sequence comparison and with Western and urban South- and Central-American isolates using multi-locus sequence analysis. A majority (77%) of the isolates carried the cancer-associated virulence gene cagA and also the s1/i1/m1 allele of the vacuolating cytotoxin gene that is linked to increased severity of disease. Moreover, it was found that Nicaraguan isolates have a blood group-binding adhesin (babA) variant highly similar to previously reported babA sequences from Latin America H. pylori isolates
11	Objective	H. pylori reference strain 26695, cagA and Cag-pathogenicity island deletion mutants were cultured	Sequencing on an Illumina MiSeq platform (Illumina)	Wong et al[105]
	Characterization of genes associated with biofilm formation in H. pylori
	Main finding
	Genes identified to be associated with biofilm formation in H. pylori included alpha (1,3)-fucosyltransferase, flagellar protein, 3 hypothetical proteins, outer membrane proteins and a Cag-pathogenicity island protein (CagPAI). These genes play a role in bacterial motility, lipopolysaccharide synthesis, Lewis antigen synthesis, adhesion and/or the type-IV secretion system (T4SS). Deletion of cagA and CagPAI confirmed that CagA and T4SS were involved in H. pylori biofilm formation
12	Objective	H. pylori was isolated from a gastroscopic antral biopsy specimen of a patient with chronic gastritis	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 150 bp)	Cao et al[155]
	H. pylori was isolated from a gastroscopic antral biopsy specimen of a 53-year-old male patient with chronic gastritis. Whole genome sequencing was applied to these isolates, and bioinformatic tools were used to investigate the within host evolution of H. pylori isolates
	Main finding
	The H. pylori genomes fall into two clades, reflecting colonization of the stomach by two distinct strains. The lineages have accumulated diversity during an estimated 2.8 and 4.2 yr of evolution. Around 150 clear recombination events between the two clades were found. Imputed ancestral sequences also showed evidence of recombination between the two strains prior to their diversification, and it was estimated that they have both been infecting the same host for at least 12 yr
13	Objective	Nineteen H. pylori clinical isolates were isolated from gastric epithelium biopsy specimens	Sequencing on an Illumina MiSeq platform (Illumina; 2 × 300 bp)	Iwamoto et al[131]
	Whole genome sequencing of 12 clarithromycin resistant and 7 susceptible H. pylori strains was done to identify novel genetic factors that reduce susceptibility to clarithromycin
	Main finding
	In clarithromycin resistant H. pylori strains specific point mutations in the 23S rRNA gene were found. In addition, genetic variants of four gene clusters (hp0605-hp0607, hp0971-hp0969, hp1327-hp1329, and hp1489-hp1487) of efflux pumps homologues, which have been previously implicated in multi-drug resistance, were found
14	Objective	H. pylori reference strain 26695 was used as amoxicillin-sensitive reference strain and as parental strain to create in vitro resistant H. pylori isolates	Sequencing on an Illumina Genome Analyzer (Illumina)	Qureshi et al[130]
	Investigation of the occurrence of genetic mutations that contribute to amoxicillin resistance in H. pylori when exposed to increasing concentrations of amoxicillin in vitro
	Main finding
	Using a whole genome sequencing approach, mutations in a number of genes were identified, notably pbp1, pbp2, hefC, hopC and hofH. Mutations in pbp1, hefC, hopC, hofH, and possibly pbp2, contributed to H. pylori high-level amoxicillin resistance
15	Objective	H. pylori reference strain 26695 and H. pylori strain J99 were grown on solid non-selective agar media	Sequencing on a PGM (Ion Torrent, Thermo Fischer Scientific, United States) and an Illumina MiSeq platform (Illumina)	Perkins et al[156]
	Next generation sequencing of the H. pylori reference strains J99 and 26695 and bioinformatic analysis of the sequencing data using publicly available algorithms. The accuracy of the coding sequence assemblies was compared to the originally published sequences
	Main finding
	With the Ion Torrent PGM, an inherently high-error rate in the raw sequencing data was found. With the Illumina MiSeq, significantly more non-covered nucleotides were found when using Illumina Nextera XT compared to the Illumina Nextera library preparation method. The most accurate de novo assemblies were found when using the Nextera technology. However, extracting an accurate multi-locus sequence type was inconsistent compared to the Ion Torrent PGM. The Cag-pathogenicity island failed to assemble onto a single contig in all technologies but was more accurate using the Nextera technology. The Illumina MiSeq Nextera method was found as the most accurate method for whole genome sequencing of H. pylori and de novo assembly of its genome

Their objective, employed sequencing method and main finding is briefly described in the table. H. pylori: Helicobacter pylori.