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Updated: Feb 19, 2026
Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants
Cristina Alcon-Giner1, Shabhonam Caim1, Suparna Mitra1,2
1The Gut Health and Food Safety Programme, Quadram Institute Bioscience, Norwich Research Park, Colney, Norwich, UK.
Insights
Optimizing DNA extraction and 16S rRNA sequencing for extremely low birth weight (ELBW) infant gut microbiota is crucial. Prolonged bead-beating and specific 16S rRNA regions (V1-V3, V6-V8) ensure accurate profiling, especially with probiotic use.
Area of Science:
- Microbiology
- Neonatal Research
- Bioinformatics
Background:
- Extremely low birth weight (ELBW) infants exhibit altered gut microbial communities.
- Microbiota disturbances in ELBW infants are linked to increased disease risk, necessitating standardized characterization methods.
- Optimizing microbiota profiling is essential for evaluating microbiota therapies like probiotics in ELBW infants.
Purpose of the Study:
- To optimize a 16S rRNA gene profiling protocol for standardizing the characterization of gut microbiota in ELBW infant fecal samples.
- To compare different DNA extraction methods and 16S rRNA gene regions for accurate microbiota profiling.
- To evaluate the impact of bioinformatics approaches on analyzing ELBW infant microbiota data.
Main Methods:
- Compared three DNA extraction methods and three 16S rRNA gene hypervariable regions (V1-V3, V4-V5, V6-V8) using ELBW infant fecal samples.
- Utilized paired shotgun metagenomics as a gold standard for validation.
- Analyzed sequencing data using two bioinformatics approaches (OTU and paired end).
Main Results:
- A prolonged bead-beating step in DNA extraction optimized bacterial DNA yield.
- 16S rRNA gene regions V1-V3 and V6-V8 provided the most representative taxonomic profiles, validated by shotgun sequencing.
- The V4-V5 region resulted in underrepresentation of certain taxa, including Bifidobacterium, and altered diversity profiles.
Conclusions:
- Optimized DNA extraction for ELBW fecal samples, especially those on probiotics, requires a prolonged bead-beating step.
- 16S rRNA regions V1-V3 and V6-V8 reliably represent ELBW infant microbiota.
- The V4-V5 region may be unsuitable for studies involving Bifidobacterium in ELBW infants.
Background:
Infants 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.
Methods:
Using 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'.
Results:
Results 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.
Conclusions:
We 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.
