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Updated: Mar 8, 2026
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Published on: December 22, 2014
Impact of sample processing on human airways microbial metagenomes
Lutz Wiehlmann1, Katarzyna Pienkowska2, Silke Hedtfeld2
1Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Core Unit 'Next Generation Sequencing', Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Institute for Human Genetics, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
Sample storage significantly impacts whole metagenome shotgun sequencing results. Immediate processing or freezing at -80°C is crucial for accurate microbial community analysis, preventing distorted metagenome profiles.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- Whole metagenome shotgun sequencing (WMS) is a powerful tool for analyzing microbial communities.
- Accurate WMS relies on unbiased sample processing, including DNA isolation and storage.
Purpose of the Study:
- To evaluate the impact of DNA isolation methods and storage duration on WMS profiles.
- To determine optimal sample handling procedures for preserving microbial community integrity.
Main Methods:
- Deep throat swabs were collected from healthy adults and an infected infant.
- DNA was isolated using sonification or enzymatic lysis immediately or after 24h storage at room temperature in Amies transport medium.
- Generated metagenomes were analyzed for changes in microbial composition and abundance.
Main Results:
- Sonification and enzymatic lysis yielded comparable high-quality metagenomes, especially for low DNA input (<10 ng).
- Storage for 24h at room temperature severely distorted metagenome profiles, leading to decreased species abundance and proliferation of specific bacteria (e.g., Streptococcus, Neisseria, Haemophilus).
- Bacterial reads increased at the expense of human reads after storage.
Conclusions:
- Sample storage duration is a critical factor influencing WMS data accuracy.
- Immediate processing or freezing at -80°C is recommended for samples intended for WMS analysis.
- Avoiding room temperature storage is essential to prevent methodology-driven bias in metagenome profiling.
