Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants

BMC Genomics

Methodology Article

Paul Clarke¹  Gusztav Belteki²  John Wain³  Cristina Alcon-Giner⁴  Jennifer Ketskemety⁴  Lindsay J. Hall⁴  Shabhonam Caim⁴  Udo Wegmann⁴  Suparna Mitra⁵ 

[1] Neonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, UK;Neonatal Intensive Care Unit, The Rosie Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK;Norwich Medical School, University of East Anglia, Norwich Research Park, Colney, Norwich, UK;The Gut Health and Food Safety Programme, Quadram Institute Bioscience, Norwich Research Park, Colney, Norwich, UK;The Gut Health and Food Safety Programme, Quadram Institute Bioscience, Norwich Research Park, Colney, Norwich, UK;Leeds Institute for Biomedical and Clinical Sciences, University of Leeds, Leeds, UK;

关键词: Extremely low birth weight infants;    Microbiota;    16S rRNA gene sequencing;    Shotgun sequencing;    DNA extraction;    Bifidobacterium;   

DOI  :  10.1186/s12864-017-4229-x

 received in 2017-08-24, accepted in 2017-10-20,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundInfants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation.MethodsUsing ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a ‘gold-standard’.ResultsResults indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level.ConclusionsWe determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member.

【 授权许可】

CC BY   
© The Author(s). 2017