title: Method optimization for fecal sample collection and fecal DNA extraction.
abstract: BACKGROUND
  This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses.
  
  
  METHODS
  Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I®, Norgen Biotek All-In-One®, MoBio PowerMag®) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures.
  
  
  RESULTS
  The optimal MSM I protocol involves a 0.2 g stool sample and 1000 μL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20°C or -80°C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/μL. The protocol was reproducible in terms of DNA yield among different stool aliquots.
  
  
  CONCLUSIONS
  We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation.
authors.affiliation: 1 Integrated BioBank of Luxemburg (IBBL) , Luxembourg .
authors.forename: Conny
authors.initials: C
authors.lastname: Mathay
authors.forename: Gael
authors.initials: G
authors.lastname: Hamot
authors.forename: Estelle
authors.initials: E
authors.lastname: Henry
authors.forename: Laura
authors.initials: L
authors.lastname: Georges
authors.forename: Camille
authors.initials: C
authors.lastname: Bellora
authors.forename: Laura
authors.initials: L
authors.lastname: Lebrun
authors.forename: Brian
authors.initials: B
authors.lastname: de Witt
authors.forename: Wim
authors.initials: W
authors.lastname: Ammerlaan
authors.forename: Anna
authors.initials: A
authors.lastname: Buschart
authors.forename: Paul
authors.initials: P
authors.lastname: Wilmes
authors.forename: Fay
authors.initials: F
authors.lastname: Betsou
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis*; D004285:Dogs; D005243:Feces / Q000737:chemistry* / Q000382:microbiology; D006801:Humans; D054892:Metagenome; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods*; D013053:Spectrophotometry
chemical_list: D004247:DNA