Gut microbiome comparability of fresh-frozen versus stabilized-frozen samples from hospitalized patients using 16S rRNA gene and shotgun metagenomic sequencing

Emma E Ilett,Gedske Daugaard,Roger Paredes,Marc Noguera-Julian,Cameron Macpherson,Jens Lundgren,Marie Helleberg,Daniel D Murray,Mette Jørgensen,Henrik Sengeløv

doi:10.1038/s41598-019-49956-7

Emma E Ilett, Gedske Daugaard + Show 8 more

Open Access

https://doi.org/10.1038/s41598-019-49956-7

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Abstract

Collection of faecal samples for microbiome analysis in acutely sick patients is logistically difficult, particularly if immediate freezing is required (i.e. fresh-frozen, or FF sampling). Previous studies in healthy/non-hospitalized volunteers have shown that chemical stabilization (i.e. stabilized-frozen, or SF sampling) allows room-temperature storage with comparable results to FF samples. To test this in a hospital setting we compared FF and SF approaches across 17 patients undergoing haematopoietic stem cell transplantation (HSCT) using both 16S rRNA gene and shotgun metagenomic sequencing. A paired (same stool specimen) comparison of FF and SF samples was made, with an overall comparable level in relative taxonomic abundances between the two sampling techniques. Though shotgun metagenomic sequencing found significant differences for certain bacterial genera (P < 0.001), these were considered minor methodological effects. Within-sample diversity of either method was not significantly different (Shannon diversity P16SrRNA = 0.68 and Pshotgun = 0.89) and we could not reject the null hypothesis that between-sample variation in FF and SF were equivalent (P16SrRNA = 0.98 and Pshotgun = 1.0). This indicates that SF samples can be used to reliably study the microbiome in acutely sick patient populations, thus creating and enabling further outcomes-based metagenomic studies on similarly valuable cohorts.

Highlights

It is becoming increasingly clear that analysis of the human gut microbiome can reveal critical aspects of human disease pathogenesis, as well as the variation of treatment responses among a variety of patient groups[1,2,3]
In our study we show that SF is comparable to freshly frozen (FF) protocols in a diseased and hospitalized cohort using both 16S rRNA gene and shotgun metagenomic sequencing data
When comparing the alpha-diversity between sampling methods we found no significant difference between SF and FF samples

Summary

Introduction

It is becoming increasingly clear that analysis of the human gut microbiome can reveal critical aspects of human disease pathogenesis, as well as the variation of treatment responses among a variety of patient groups[1,2,3]. As bacterial species can differ in diseased populations, it is essential to collect this information and evaluate whether SF affects their compositions or not To address these remaining aspects of collection-associated bias within gut microbiomes of the diseased, we have conducted a study comparing FF samples to those collected using a SF protocol. This is the first study (to our knowledge) assessing the differences between these protocols using both 16S rRNA gene and shotgun metagenomic sequencing in a hospital setting. HSCT serves as a useful disease proxy as it is reserved for severely diseased patients and, critically, has been shown to heavily impact microbiome structure[24]

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