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Jennifer Nagel. A short summary of this paper. R8 :8Y8! SM: 8! V9FS 8Q? K:8 QL! LMFN Q! SFN Q! R8O :9?! GF 8GH! LHF 89! JFLL:R 8! V: 8Q! GFIJ K! LF :Q!? JNjj 8! M8 JL! GF9O L:G! Q: GN H:8L! L8HH 8Q! R8 :8Y8Q! GFN Q! R FGV! FNH L! G NQ:9?! Q: GN H! R8 F9? Y:O tY89N8! LNJJ :8Q! GF9O 89FN?

H8 :9? IN 8l! GF NI9! GF NI9T! N9 8LL! Q:LLF Y8! Two modes of cognition 89H K! JLKO U! GMF F? LJ :H! N9 :V8 K! GF9O J89! LHF 89T! LJ8 8Q! R8 FS! NGS platforms are now increasingly used in the field of clinical research [ 2 ]. Metagenomic sequencing offers numerous advantages over traditional culture-based techniques that have long been the standard test for detecting pathogenic bacteria. This method is particularly useful for characterizing uncultured bacteria and novel pathogens [ 3 ].

Among sequence-based bacterial analyses, amplicon sequencing of the 16S ribosomal RNA rRNA gene has proven to be a reliable and efficient option for taxonomic classification [ 4 , 5 ].

The bacterial 16S rRNA gene contains nine variable regions V1 to V9 that are separated by highly conserved sequences across different taxa. For bacterial identification, the 16S rRNA gene is first amplified by polymerase chain reaction PCR with primers annealing to conserved regions and then sequenced. The sequencing data are subjected to bioinformatic analysis in which the variable regions are used to discriminate between bacterial taxa [ 6 ].

Since the conventional parallel-type short-read sequencer cannot yield reads covering the full length of the 16S rRNA gene [ 7 ], several regions of it have been targeted for sequencing, which often causes ambiguity in taxonomic classification [ 8 ]. New sequencing platforms have overcome these technical restrictions, particularly those affecting read length. This is probably due to sequence mismatches in the primer used for 16S rRNA gene amplification [ 20 ].

Deviations or aberrancies in the Bifidobacterium composition in the human gut have been reported in several diseases including obesity, allergy, and inflammatory disorders [ 21 ].

Based on their putative health-promoting effects, several strains of Bifidobacterium have been utilized as probiotics [ 22 ]. Within these contexts, the species-level characterization of Bifidobacterium diversity in human gut microbiota is potentially important in clinical practice.

Its applicability to highly complex bacterial communities has not yet been thoroughly investigated. S1a , which biases amplification toward underrepresentation of Bifidobacterium species Additional File 2 : Supplementary Fig. To overcome this drawback, we introduced three degenerate bases to the 16S rRNA gene-specific sequences of the primer Additional File 2 : Supplementary Fig. The competence of the modified primer set was then evaluated by 16S rRNA gene sequence analysis of a ten-species mock community.

Following adapter trimming and size selection, reads Randomly sampled reads were aligned against our in-house genome database GenomeSync [ 18 ]. Full-length 16S rRNA gene amplicon sequencing with the modified primer set led to the successful identification of all expected bacterial genera, including Bifidobacterium Fig.

S3a, S3b. The majority of reads were correctly classified down to the species level, demonstrating the excellent discriminatory power of the full-length sequencing method for bacterial identification Fig. Bacillus species was an exception in the analysis with both the GenomeSync and NCBI reference database the SILVA database does not include species level information , and discrimination of Bacillus cereus from the closely related species such as Bacillus anthracis and Bacillus thuringiensis was not achieved Additional File 4 , Additional File 6.

Likewise, Escherichia coli was not reliably distinguished from Shigella and other Escherichia species sharing the high 16S rRNA gene sequence similarity to each other [ 25 , 26 ], and species-level resolution was not possible.

Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction.

Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of representative bacterial species. The pie charts represent taxonomic profiles at the b genus and c species levels. Even with the full-length 16S rRNA gene analysis, species-level resolution is not possible for Bacillus and Escherichia.

Slices corresponding to misclassified assigned to bacteria not present in the mock community or unclassified not classified at the given level but placed in a higher taxonomic rank reads are exploded. Min: minimum read length, Avg: average read length, Max: maximum read length, Q score: average Phred quality score. The percentage of reads retained after size filtering is shown in parentheses. We compared the resolution of full-length and short-read 16S rRNA gene amplicon sequencing for the taxonomic classification of bacteria.

In contrast to full-length sequencing with the highest resolution, a significant number of V3-V4 reads were misclassified or assigned to a higher taxonomic rank Fig. The three alignment tools worked with some differences in assigning the V3-V4 sequences. This was notable for alignments against the GenomeSync database, where most V3-V4 reads derived from Enterococcus faecalis and Escherichia coli were not correctly assigned to each taxon, as more than one species produced the same similarity score for the sequence read queries and the reads were ranked at the lowest common ancestor Additional File 3 : Supplementary Table S5, Additional File 7.

We could not classify these bacteria even at the phylum level. The results suggest an analytical problem such as database errors, which may give rise to assigning a distantly related organism to the query sequences. The classifications were not affected by increasing the number of analyzed reads to 10, Additional File 2 : Supplementary Fig.

These classification problems were solved, for the most part, by the V1-V9 long-read sequencing. Thus, regardless of program and database used, the full-length 16S rRNA gene sequencing appeared to give better resolution for bacterial identification. V3-V4 short-read sequencing showed a discriminatory power comparable to that of V1-V9 full-length sequencing in the classification of Deinococcus , Rhodobacter , and Streptococcus. However, the V3-V4 region was not suitable for species-level identification of some genera such as Clostridium and Staphylococcus.

These results suggest a lower resolution of the V3-V4 region for species-level classification, emphasizing the advantage of long-read sequencing for obtaining an accurate representation of the sample bacterial composition. Classification accuracy compared between full-length V1-V9 and partial V3-V4 16S rRNA gene sequencing data obtained from composition profiling of the ten-species mock community.

The donut charts show the proportions of reads correctly assigned to the species constituting the mock community. The percentage of correctly classified reads is shown in the center hole. ND: not determined species-level resolution is not possible for Escherichia.

We assessed the performance of our full-length 16S rRNA gene amplicon sequencing approach in the context of a highly complex bacterial community. Table 2. The reads were mapped against the GenomeSync reference database for taxonomic assignment. In Fig. The curve started to plateau at around 20, reads.

Based on these observations, randomly sampled 20, reads were used in further analysis to determine the bacterial composition of the human gut. Numbers of detected species are plotted against numbers of reads used for taxonomic classification. S6, Additional File 12 [ 27 , 28 ]. This result confirmed the validity of our method for the taxonomic classification of the bacterial community. Statistically significant similarities have been found in the relative genus abundances across these sequencing methods.

Comparison of taxonomic profiles of human gut microbiota between sequencing methodologies. Randomly sampled 20, reads from each data set were allocated to the reference genome database of representative bacterial species. The 15 most abundant taxa are shown.

The Pearson correlation coefficient r between sequencing methods was computed. While genus classification using long versus short reads was relatively comparable, we observed considerable differences across amplified regions in the species-level profiling of human gut microbiota.

As shown in Fig. The poorer read quality gave rise to assigning multiple species to a query sequence, leading to the increased number of reads not classified at the species level Additional File Comparison of taxonomic resolution. Horizontal bars represent mean values. For Bifidobacterium , there appeared to be considerable deviations in the relative species abundances depending on the sequencing method used Fig.

A significant number of the V3-V4 reads, however, were assigned erroneously to Bifidobacterium species of non-human origin in the direct read mapping approach using the relatively shallow GenomeSync reference database Additional File 2 : Supplementary Fig.

Except for Bifidobacterium longum , Bifidobacterium species could not be reliably identified by the V3-V4 sequencing strategy and they were ranked at the genus level Additional File 2 : Supplementary Fig. Species composition of Bifidobacterium in six fecal samples. Results obtained by the three sequencing methods are shown. The legends show the 14 most abundant Bifidobacterium species.

Instead, the reads were analyzed by the direct read mapping method that assigns sequences to taxonomic bins based on the similarity to a reference database [14, 15]. For improving the classification results, the reads were filtered by length to eliminate those outside the expected size range. Typically, extremely short reads possess only one primer-binding site, suggesting that they are derived from incomplete sequencing. There also exist unexpectedly longer reads with a continuous sequence structure in which two 16S rRNA gene amplicons are linked end-to-end.

Because these reads can potentially result in unclassified reads or misclassification, they were eliminated before alignment to the reference sequences of the bacterial genome. This method can be applied to a wide range of sequence-based analyses, including detection of functional genetic markers like antimicrobial resistance genes and identification of genetic variations in targeted loci [ 11 , 34 , 35 ].

The full-length 16S rRNA gene sequencing provided better resolution than short-read sequencing for discriminating between members of certain bacterial taxa, including Bifidobacterium , Clostridium , Enterococcus , and Staphylococcus. Consistently, comprehensive microbiome studies using a sequencing data set consisting of different regions of the 16S rRNA gene have shown that the choice of the regions to be sequenced substantially affects the classification results, and some bacterial species are identified only by sequencing the entire 16S rRNA gene [ 6 , 36 , 37 ].

It is important to note, however, that even full-length 16S rRNA gene analysis fails to discriminate some closely related species such as members of Bacillus cereus group and Escherichia , indicating the limitations of the 16S rRNA gene amplicon sequencing alone in species allocation. Long read sequencing targeting other phylogenetic markers may be an alternative to 16S rRNA gene amplicon sequencing and provide better resolution for bacterial identification.

Our study has some limitations. Duty 13 Represent the external face of the business, exhibiting the expected corporate image and quality service standards to customers. Duty 14 Process goods in line with agreed procedures. For example, refusals, returns or partial deliveries. Duty 15 Complete any additional contracted services, using the correct tools and equipment and providing product information.

This includes for instance assembly, installation, packing and positioning. Duty 16 Adjust customer contracts within limits of own role, escalating issues where required. Duty 17 Complete all required reporting procedures in accordance with the contract and own organisations procedures and formats e.

Duty 18 Follow continuous professional development, maintaining own drivers licence and ensuring required training and knowledge is kept up to date. K18 K20 K23 K24 K K1 : Urban vehicle preparation and maintenance requirements, within limits of own role. Back to Duty. K2 : Different types of goods transported by fixed axle vehicles over kg in weight. K3 : Personal protective equipment selection and use.

K4 : The principles of load and weight distribution applicable to fixed axle vehicles over kg in weight. K5 : Mechanical and manual handling techniques when using auxiliary equipment. For example, using a mechanical grab for waste collection. K6 : The capability and limitations of fixed axle vehicles over kg in weight. This includes manoeuvrability, space requirements, access requirements, legal restrictions and physical constraints.

K7 : The regulations and legislation that impact on professional driving. K8 : The highway code road laws and road restrictions applicable to category C and C1 licence holders. K9 : A range of driving techniques applicable to fixed axle vehicles over kg in weight. K10 : Methods to counteract for road and weather conditions impacting fixed axle vehicles over kg in weight.

K11 : The features found in urban environments including congestion charging, street furniture, pedestrians, and other road users. K12 : Map reading techniques relating to the UK road network and urban environments.

K13 : Hazard perception techniques when driving, including the different approaches used in urban areas and other UK road environments. K14 : Accident reporting and incident management. K15 : Environmental and sustainability factors when driving in urban areas.

K16 : Techniques for protecting goods in urban areas, including both when in transit and when the vehicle is unattended. K17 : Uses and limitations of urban vehicle in-cab technology for safety, reporting and compliance.

This includes driver aids, telematics, handheld terminals, and on-board weighing systems. K18 : The importance of brand identity in the urban delivery sector. K19 : The role of customer services standards in urban delivery, including the impact that own service provision can have on both the customer and on the wider organisation. K20 : Different forms of communication. For example, electronic, written and in-person. K21 : A range of dynamic risk assessment methods and associated reporting.

K22 : The different regulations and legislation that apply when working on-site. For example, compliance and health and safety requirements in yards, businesses, and homes.

K23 : Techniques for managing own well-being physical and mental health in an urban delivery environment. K24 : The different types of organisation that make up the urban delivery supply chain. K25 : The range and applications of own organisations products and on-site services. S1 : Prepare a fixed axle vehicle over kg in weight for the planned daily workload. This includes the cab, fluid levels, and general inspection.

S2 : Monitor charge or fuel level of the vehicle to meet the daily requirements of the urban schedule Back to Duty.

S3 : Monitor the vehicle for defects. S4 : Co-ordinate own work with others to meet business priorities. S5 : Apply protections, manual handling and mechanical aids to the situation, when loading or off-loading goods. S6 : Prepare, position and secure goods appropriate for the goods type, the vehicle type and the urban conditions.

S7 : Manage goods in transit. For example, security, and checking seals for signs of damage and leaks. For example single and multiple urban deliveries or collections. S9 : Drive fixed axle vehicles over kg in weight considering all relevant factors. This includes vehicle type, road surface, goods, environmental conditions, vulnerable road users and pedestrians Back to Duty. S10 : Use on-board electronic systems in line with operating procedures.

S11 : Respond and adapt to urban driving incidents, accidents roadworks and hazards. S12 : Manoeuvre fixed axle vehicles over kg in weight on site. For example, a customer driveway, building site, or recycling centre. S13 : Perform dynamic risk assessment of a site and take remedial action.

For example, adjusting or aborting a delivery or collection due to safety issues. S14 : Manage relationships that enable successful urban delivery, collection and on-site contracts. S15 : Adapt communication style to meet the needs of the audience. S17 : Complete contractual obligations on site.

For example, installing white goods in a home, or removing waste, leaving the site to the expected standard. S18 : Brief the customer on the technical specifications of the delivery, collection or installation, answering questions. This could mean, for instance, demonstrating how a product works. S19 : Adjust the services provided in response to customer requirements, within the limits of own role. S20 : Comply with relevant legislation and regulation, both when driving and on site Back to Duty.

B1 : Work flexibly for example, working alone and in a team as required.