Amos, G

Bajaj-Elliott, M

Gerasimidis, K

Gut Microbiota for Health expert panel of British Society of Gas

Hansen, R

Hold, GL

Iqbal, TH

Marchesi, JR

Thomas, LV

English

UCL Discovery

Title: Next-generation sequencing as a clinical laboratory tool for describing different microbiotas: an urgent need for future paediatric practice  Short Title: Next-generation sequencing different microbiotas in paediatrics  Authors: Richard Hansen1, Mona Bajaj-Elliott2, Georgina L. Hold3, Konstantinos Gerasimidis4, Tariq H. Iqbal5, Gregory Amos6, Linda V. Thomas7, Julian R. Marchesi8. On behalf of Gut Microbiota for Health expert panel of British Society of Gastroenterology.  Affiliations: 1. Royal Hospital for Children, Paediatric Gastroenterology, Glasgow, United Kingdom  2. Department of Infection, Immunity and Inflammation, Institute of Child Health, University College London, London, United Kingdom 3. Microbiome Research Centre, St George and Sutherland Clinical School, University of New South Wales, Sydney, Australia 4. Human Nutrition, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom 5. Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom 6. National Institute for Biological Standards and Control, South Mimms, United Kingdom  7. Gut Microbiota for Health expert panel, British Society of Gastroenterology, United Kingdom 8. Department of Metabolism, Digestion and Reproduction, Imperial College London, London, United Kingdom  Licence Statement: I, the Submitting Author has the right to grant and does grant on behalf of all authors of the Work (as defined in the below author licence), an exclusive licence and/or a non-exclusive licence for contributions from authors who are: i) UK Crown employees; ii) where BMJ has agreed a CC-BY licence shall apply, and/or iii) in accordance with the terms applicable for US Federal Government officers or employees acting as part of their official duties; on a worldwide, perpetual, irrevocable, royalty-free basis to BMJ Publishing Group Ltd (“BMJ”) its licensees and where the relevant Journal is co-owned by BMJ to the co-owners of the Journal, to publish the Work in Archives of Disease in Childhood and any other BMJ products and to exploit all rights, as set out in our licence. The Submitting Author accepts and understands that any supply made under these terms is made by BMJ to the Submitting Author unless you are acting as an employee on behalf of your employer or a postgraduate student of an affiliated institution which is paying any applicable article publishing charge (“APC”) for Open Access articles. Where the Submitting Author wishes to make the Work available on an Open Access basis (and intends to pay the relevant APC), the terms of reuse of such Open Access shall be governed by a Creative Commons licence – details of these licences and which Creative Commons licence will apply to this Work are set out in our licence referred to above.  Corresponding Author: Richard Hansen, Royal Hospital for Children, 1345 Govan Road, Glasgow; richard.hansen@glasgow.ac.uk; Tel: +44 141 451 6543  Word Count: 498  Acknowledgements: RH is supported by a Career Researcher Fellowship from NHS Research Scotland. JRM is supported by the NIHR Imperial Biomedical Research Centre.  Competing Interests: RH has received consultancy fees and conference travel support from 4D Pharma and Nutricia. GLH has received consultancy fees and conference travel support from Ferring and Proctor & Gamble. GLH is also a member of Enterobiotix scientific advisory panel. KG has received research grants, speaker’s and consultancy fees from Nestle Health Science, Nutricia-Danone, Mylan, Abbott and Baxter. THI has received speaker’s fees from Vifor, Pharmacosmosos, Shield, Takeda, Roche and Ferring. JRM has received consultancy fees from Enterobiotix.  Authors Contributions: All authors contributed equally.  Key Words: Microbiology Paediatrics Microbiome  Letter Text: We read with interest the editorial by Baralle and Ismail exploring the utility of next-generation sequencing in paediatric clinical genetics1. The other major use of this technology, which has emerged over the last decade, is in cataloguing complex microbial communities, for instance the different human microbiomes. The microbiome describes the diverse range of microorganisms found in and on the human body and their complex interaction network. It is generally considered as comprising viruses, bacteria, archaea and fungi. The human gut contains the most complex microbial ecosystem and has been most extensively investigated to date, particularly the gut bacterial microbiota.  Briefly, two main approaches are used to identify and describe bacterial communities using next-generation sequencing: amplicon sequencing (metataxonomics) and metagenomics2. Amplicon sequencing relies on PCR amplification of a single bacterial “housekeeping” gene (the 16S rRNA gene) which is considered ubiquitous but helpfully contains both highly conserved and variable regions across species. Comparing the DNA sequence read of a section of the gene against a reference database, particularly the variable regions, allows identification of the corresponding bacterium to species level if sufficient sequence data is analysed. Next-generation sequencing allows the reading of many thousands of amplicons in parallel, allowing for its use in complex microbial communities. In contrast, metagenomic sequencing reads all the DNA present in a sample after it has been randomly broken down into fragments, hence its other name- “shotgun sequencing”. Amplicon sequencing has the benefit of focussing on bacterial information but relies on inferring genomic content data from elsewhere. Metagenomics is more comprehensive but difficult to implement in mixed source DNA samples, particularly gut mucosal biopsies where human DNA far outweighs the microbial component, because all DNA sources are sequenced together by this approach. Metagenomics also suffers from annotation problems, with a large proportion of sequence reads remaining “unknown” after analysis. Novel studies of the gut microbiome in the paediatric population have described new microbial diagnostic and prognostic markers, and have opened novel therapeutic avenues. For example, a reduction in 11 different bacteria in faeces from children with coeliac disease are specific to that disease state3; a distinct faecal bacterial cluster described in children post-allogenic haematopoietic stem cell transplantation confers distinct prognostic outcomes in terms of subsequent viraemia episodes (unpublished data from author MBE); and exploration of microbiota changes during exclusive enteral nutrition treatment for Crohn’s disease4 has led directly to the development of new solid food approaches to treating the condition5. If we are to embrace the diagnostic, prognostic and therapeutic revolution offered to us by a greater understanding of the microbiome, there is an urgent need to integrate microbiome analysis into clinical practice. To our knowledge, no UK centre currently offers this in an accredited clinical laboratory. Whilst there are significant financial, logistical, methodological, analytical and scientific challenges to undertaking clinical microbiota testing, it is surely time for the scientific microbiome community to move these techniques from the academic laboratory into the clinical setting, much as our colleagues in clinical genetics have already successfully achieved.  References: 1. Baralle D., Ismail V. “Next Generation Sequencing” as a diagnostic tool in paediatrics. Arch Dis Child 2021;106(1):1–2. Doi: 10.1136/archdischild-2020-320251. 2. Clegg F., Berry SH., Hansen R., Hold GL. Molecular analysis of the microbiome in colorectal cancer. In: Beaulieu J-F, editor. Colorectal Cancer: Methods and Protocols, Methods in Molecular Biology. Springer; 2018. p. 139–53. 3. Zafeiropoulou K., Nichols B., Mackinder M., Biskou O., Rizou E., Karanikolou A., et al. Alterations in Intestinal Microbiota of Children With Celiac Disease at the Time of Diagnosis and on a Gluten-free Diet. Gastroenterology 2020;159(6):2039-2051.e20. Doi: 10.1053/j.gastro.2020.08.007. 4. Quince C., Ijaz UZ., Loman N., Eren AM., Saulnier D., Russell J., et al. Extensive modulation of the fecal metagenome in children with Crohn’s disease during exclusive enteral nutrition. Am J Gastroenterol 2015;110(12):1718–29. Doi: 10.1038/ajg.2015.357. 5. Svolos V., Hansen R., Nichols B., Quince C., Ijaz UZUZ., Papadopoulou RTRT., et al. Treatment of active Crohn’s disease with an ordinary food-based diet that replicates exclusive enteral nutrition. Gastroenterology 2019;156(5):1354–67. Doi: 10.1053/j.gastro.2018.12.002. 

Next-generation sequencing as a clinical laboratory tool for describing different microbiotas: an urgent need for future paediatric practice.

https://discovery.ucl.ac.uk/10125470/3/Bajaj-Elliott_Next-generation%20sequencing%20different%20microbiotas%20in%20paediatrics.pdf

Next-generation sequencing as a clinical laboratory tool for describing different microbiotas: an urgent need for future paediatric practice.

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