P851 Effects of conservation time on the diversity and relative abundance of intestinal microbiome in samples of faecal matters
M. León F1,2, C.A. Nieto2, Z. Corredor2, C. Flórez-Sarmiento3, V. Parra-Izquierdo3,4, C. Romero-Sanchez1,4
1Universidad El Bosque, Cellular and Molecular Immunology Group/ INMUBO, Bogota D.C, Colombia, 2Universdidad El Bosque, Bacterial Molecular Genetics Laboratory, Bogota D.C, Colombia, 3Grastroadvanced SAS, Gastroenterology, Bogota D.C, Colombia, 4Hospital Militar Central/Universidad Militar Nueva Granada, Rheumatology and Immunology Department- School of Medicine- Clinical Immunology Group, Bogota D.C, Colombia
Background
Given the complexity and diversity of the intestinal microbiome, there is a technical challenge of finding the best way to study the factors that affect the quality of the sample to obtain results in a precise an complete way.
Methods
To establish the effect of storage time under constant cryopreservation conditions (−20°C) in stool samples for the study of the gastrointestinal microbiome
Results
A sample of stool was distributed in 3 fractions (1, 2 and 3). Time 0 without cryopreservation and immediate DNA extraction (1). The samples of 15 (2) and 30 (3) days were cryopreserved at −20°C before the extraction of the genetic material. After ultracentrifugation at 4 degrees Celsius, the precipitate was subjected to enzymatic and mechanical lysis to obtain the total DNA. The DNA quality was evaluated techniques to ensure the quality and concentration of genetic material. Once the DNA of the three samples was obtained, they were subjected to the latest generation of a variable region of the 16S gene, using MiSeq technology (Illuminates). Data in relative abundances and frequencies were recorded. Project supported by the Administrative Department of Science, Technology and Innovation-Colciencias Health Call 2017, Code 130877757442
Conclusion
About 140 thousand readings were obtained for samples 1 (day 0) and 2 (day 15) and 160 thousand for sample 3 (day 30), with a reading quality Q20% between 97 and 98 and Q30% around 94, indicating a high-reliability value for each of the samples. Regarding the classification from the Operating Taxonomic Unit (OTU), 119, 99 and 106 were reported for samples 1, 2 and 3, respectively. The data obtained revealed changes over time at the level of the main edges reported for normal microbiome (Bacteroidetes, Firmicutes, Actinobacteria) between 1 and 2 vs. sample 3. For Firmicutes and Actinobacteria, decreases in abundance of 34% were observed (samples 1 and 2) at 20% for Firmicutes in sample 3 and from 4% (samples 1 and 2) to 0.8% (sample 3) for Actinobacteria. On the contrary, Bacteroidetes presented an increase in its abundance from 58% (Samples 1 and 2) to 76% (sample 3). The Proteobacterium edge did not show significant changes in its abundance
It was possible to demonstrate that the cryopreservation time before DNA extraction is an important variable that influences the percentage and integrity of the microbiota from faecal matter. Confirming that the maximum reliable shelf life at −20° in stool samples is up to 15 days, suggesting that for longer storage the temperature decrease up to −80° should be taken into account to maintain stability in the results of relative abundance and bacterial diversity