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

Inter-individual differences in faecal microbiome composition consistently outweighed any effects introduced by freeze-thaw cycles.

• Study used 16S rRNA gene sequencing to assess microbiome composition across repeated freeze-thaw cycles
• Beta diversity analyses demonstrated that sample identity (inter-individual variation) was the dominant source of variation rather than freeze-thaw treatment
• This pattern was consistent across all freeze-thaw cycles examined (FT1 through FT6)

Differential abundance analysis with MaAsLin2 identified a limited number of significantly altered microbial genera after the first freeze-thaw cycle compared to fresh, unfrozen material.

• Comparison was made between fresh unfrozen samples and samples subjected to the first freeze-thaw (FT1) cycle
• Only a limited number of microbial genera showed significant changes at FT1
• The second and third freeze-thaw cycles showed no significant changes compared to the first freeze-thaw cycle
• Subsequent cycles four through six showed minor but progressive shifts in specific taxa

The more conservative differential abundance method ALDEx2 showed no significant changes in microbial composition across any freeze-thaw cycle.

• ALDEx2 was applied as a more conservative statistical method compared to MaAsLin2
• No significant changes were detected across any of the freeze-thaw cycles (FT1 through FT6) using ALDEx2
• The discrepancy between MaAsLin2 and ALDEx2 results suggests that changes identified by MaAsLin2 may be at the margins of statistical significance

Repeated freeze-thaw cycles beyond the first showed a pattern of minor but progressive compositional shifts in specific taxa from cycles four to six.

• FT2 and FT3 showed no significant changes relative to FT1
• FT4 through FT6 showed minor but progressive shifts in specific taxa when assessed with MaAsLin2
• These progressive shifts were not confirmed as significant by ALDEx2 analysis

The study supports the reuse of stored faecal samples that have undergone a single thaw for 16S rRNA gene sequencing-based microbiome analyses.

• Reanalysing previously collected samples was investigated as a time-efficient alternative to initiating new cohort studies
• Findings indicate minimal risk of compromising microbiome integrity following a single thaw
• The study addressed concerns that repeated freeze-thaw cycles may introduce bias in downstream microbiome analyses