title: Processing faecal samples: a step forward for standards in microbial community analysis.
abstract: BACKGROUND
  The microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases in diarrhoeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis.
  
  
  RESULTS
  We collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N = 8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N = 4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples.
  
  
  CONCLUSIONS
  The degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium.
authors.forename: Alba
authors.initials: A
authors.lastname: Santiago
authors.forename: Suchita
authors.initials: S
authors.lastname: Panda
authors.forename: Griet
authors.initials: G
authors.lastname: Mengels
authors.forename: Xavier
authors.initials: X
authors.lastname: Martinez
authors.forename: Fernando
authors.initials: F
authors.lastname: Azpiroz
authors.forename: Joel
authors.initials: J
authors.lastname: Dore
authors.forename: Francisco
authors.initials: F
authors.lastname: Guarner
authors.affiliation: Vall d'Hebron Research Institute, Digestive System Research Unit, Passeig de la Vall d'Hebron 119-129, Barcelona 08035, Spain. cmanicha@gmail.com.
authors.forename: Chaysavanh
authors.initials: C
authors.lastname: Manichanh
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D058448:Biota; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D064368:Healthy Volunteers; D006801:Humans; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013048:Specimen Handling / Q000379:methods* / Q000592:standards*
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S