Latitude in sample handling and storage for infant faecal microbiota studies: the elephant in the room?

Abstract

BackgroundIn this manuscript, we investigate the “stones best left unturned” of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities by the 16S ribosomal ribonucleic acid (rRNA) gene.We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a “normal” degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities.ResultsExtraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at −80 °C prior to extraction. After 2 years, we see relatively few changes: increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after 2 days. Mailing of samples during this time period (a common form of sample collection from outpatients for example) does not lead to any additional variation.ConclusionsImportant methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at −80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at −80 °C or extracted within 2 days if stored at room temperature, with mail samples being mailed on the day of collection.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0186-x) contains supplementary material, which is available to authorized users.

Reducing Viability Bias in Analysis of Gut Microbiota in Preterm Infants at Risk of NEC and Sepsis

“…Sample storage had an insignificant effect on observed Bray–Curtis community similarity between treatment conditions (Table 3), and no separation by PMA-treatment within storage groups was observed (Figure S3). This data further supports Shaw et al (2016), findings suggesting freeze storage of severely preterm stool samples does not significantly impact the gut microbiota observed with or without PMA-treatment.…”

Consensus on standard biobanking of gut microbiota

“…(b) Transportation of stool samples. The specimens should be immediately cryopreserved at −80 °C, which is the gold standard for storing fresh stool samples . An anaerobic environment should be sustained in the storage device and the anaerobic device should not be in direct contact with the stool samples .…”

“…Each specimen should weigh no less than 200 mg (after the removal of food residue). A sample weighing 25 mg may not affect DNA extraction and sequencing but needs to be carefully selected …”

“…The specimens should be immediately cryopreserved at −80 C, which is the gold standard for storing fresh stool samples. [21][22][23] An anaerobic environment should be sustained in the storage device 24,25 and the anaerobic device should not be in direct contact with the stool samples. 3 To ensure the accuracy of the test results the sample should be frozen or stored in a sealed container with preservation solution within 15 minutes of being collected.…”

Insights into study design and statistical analyses in translational microbiome studies

“…A large body of work in the field has addressed day-to-day operations (38) involved in performing microbiome studies in research and epidemiological settings (39,40) including sample collection and storage (41)(42)(43), laboratory protocols for sample processing (40,44), choice of microbiome sequencing protocols (45) and the computational infrastructure required for data processing and storage (46,47). This review builds on these discussions while specifically focusing on the design of translational microbiome studies and development of downstream statistical analysis plans which answer complex questions in a translational setting and inform early and late-phase clinical trials.…”