title: Comparison of stool versus rectal swab samples and storage conditions on bacterial community profiles.
abstract: Sample collection for gut microbiota analysis from in-patients can be challenging. Collection method and storage conditions are potential sources of variability. In this study, we compared the bacterial microbiota from stool stored under different conditions, as well as stool and swab samples, to assess differences due to sample storage conditions and collection method.
  
  
  
  Using bacterial 16S rRNA gene sequence analysis, we compared the microbiota profiles of stool samples stored and collected under various conditions. Stool samples (2 liquid, 1 formed) from three different patients at two hospitals were each evaluated under the following conditions: immediately frozen at -80°C, stored at 4°C for 12-48 hours before freezing at -80°C and stored at -20°C with 1-2 thaw cycles before storage at -80°C. Additionally, 8 stool and 30 rectal swab samples were collected from 8 in-patients at one hospital. Microbiota differences were assessed using the Yue and Clayton dissimilarity index (θYCdistance) and analysis of molecular variance (AMOVA).
  
  
  
  Regardless of the storage conditions, the bacterial communities of aliquots from the same stool samples were very similar based on θYCdistances (median intra-sample θYCdistance: 0.035, IQR: 0.015-0.061) compared to aliquots from different stool samples (median inter-sample θYCdistance: 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001). For the stool and rectal swab comparison, samples from different patients, regardless of sample collection method, were significantly different (AMOVA p-values: <0.001-0.029) compared to no significant difference between all stool and swab samples (AMOVA p-value: 0.976). The θYCdissimilarity index between swab and stool samples was significantly lower within individuals (median 0.17, IQR: 0.10-0.27) than between individuals (median 0.93, IQR: 0.85-0.97) (Wilcoxon test p-value: <0.0001), indicating minimal differences between stool and swab samples collected from the same individual over the sampling period.
  
  
  
  For gastrointestinal microbiota studies based on bacterial 16S rRNA gene sequence analysis, interim stool sample storage at 4 °C or -20 °C, rather than immediate storage at -80 °C, does not significantly alter results. Additionally, stool and rectal swab microbiotas from the same subject were highly similar, indicating that these sampling methods could be used interchangeably to assess the community structure of the distal GI tract.
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, MI, 48109, USA. cbassis@umich.edu.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Bassis
authors.affiliation: Department of Medical Laboratory Science, Rush University Medical Center, Chicago, IL, 60612, USA.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Moore
authors.affiliation: Department of Medicine (Infectious Diseases), Rush University Medical Center, Chicago, IL, 60612, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Lolans
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Seekatz
authors.affiliation: Department of Medicine (Infectious Diseases), Rush University Medical Center, Chicago, IL, 60612, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Weinstein
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Vincent B
authors.initials: VB
authors.lastname: Young
authors.affiliation: Department of Medicine (Infectious Diseases), Rush University Medical Center, Chicago, IL, 60612, USA.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Hayden
mesh_terms: D000704:Analysis of Variance; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D002641:Chicago; D004269:DNA, Bacterial / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005260:Female; D000069196:Gastrointestinal Microbiome / Q000235:genetics; D041981:Gastrointestinal Tract / Q000382:microbiology; D006761:Hospitals; D006801:Humans; D008297:Male; D008875:Middle Aged; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA gene sequences; Gastrointestinal tract; Gut microbiota; Microbial community; Microbiota; Rectal swab; Stool