# Effects of repeated freeze and thaw cycles on the stability of faecal microbiome composition

## TL;DR

This study shows that repeated freeze-thaw cycles have minimal impact on the stability of the faecal microbiome, supporting the reuse of stored samples for research.

## Contribution

The study provides empirical evidence that freeze-thaw cycles minimally affect microbiome composition, enabling the reuse of stored faecal samples.

## Key findings

- Inter-individual differences in microbiome composition are much larger than those caused by freeze-thaw cycles.
- Only minor and progressive shifts in specific taxa were observed after multiple freeze-thaw cycles.
- Conservative statistical methods found no significant changes in microbiome composition across freeze-thaw cycles.

## Abstract

Reanalysing previously collected samples to address new research questions offers a time-efficient alternative to initiating new cohort studies. However, concerns remain regarding the stability of the faecal microbiome following repeated freeze–thaw (FT) cycles, which may introduce bias in downstream analyses. To this end, we investigated the effect repeated FT cycles had on 16 S rRNA gene sequencing and its ability to accurately measure the gut microbiome composition consistently. Our analysis showed that inter-individual sample differences consistently outweighed any effects introduced by FT. Differential abundance analysis with MaAsLin2 identified a limited number of significantly altered microbial genera after the first FT, when compared to the fresh, unfrozen material. The following second and third FT cycles showed no significant changes compared to the first FT cycle. Subsequent cycles (four to six) showed minor but progressive shifts in specific taxa. However, analysis with the more conservative method ALDEx2 showed no significant changes across any FT cycle. These findings support the reuse of stored faecal samples that have undergone a single thaw, suggesting minimal risk of compromising microbiome integrity.

The online version contains supplementary material available at 10.1038/s41598-026-39939-w.