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Methodology challenges in studying human gut microbiota - effects of collection, storage, DNA extraction and next generation sequencing technologies.

Author(s): Panek M, Čipčić Paljetak H, Barešić A, Perić M, Matijašić M, Lojkić I, Vranešić Bender D, Krznarić Ž, Verbanac D

Publication: Sci Rep, 2018, Vol. 8, Page 5143

PubMed ID: 29572539 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of fecal specimen collection, specimen storage protocols, and bacterial DNA extraction methods on DNA yield and quality (A260/A280) and on the microbial profiles obtained using two different NGS platforms. Specimens were collected from four healthy participants (age range: 33–42 years, 2 males and 2 females) in a sterile container (fresh) and with the OMNIgene.GUT Kit. Fresh specimens were aliquoted (1-2 g) and one aliquot was processed within a few hours of collection while the remaining aliquot was frozen at -20°C for 14 days. Specimens collected with the OMNIgene.GUT Kit were aliquoted (400-500 mg) and one aliquot was processed within a few hours of collection and the other was stored at room temperature for 14 days. DNA was extracted from each aliquot using the MP Biomedicals Fast DNA SPIN Kit for Feces, the QIAgen QIAamp Fast DNA Stool Mini Kit, and the MO BIO Power Fecal DNA Isolation Kit. DNA quantity and quality (A260/A280) were determined by spectrophotometry (Nanodrop 2000) and fluorometry (Qubit 3.0). Fecal microbial profiles were analyzed by amplification of the 16S rRNA gene using the Nextera XT DNA Sample Preparation Kit and sequencing on the Ilumina MiSeq and using the 16S Ion Metagenomics Kit and sequencing on the Ion Torrent PGM platform. Alpha diversity was assessed using observed OTUs, Chao1 index, and PD_whole_tree index. Differences in beta diversity were presented as principal coordinate analysis plots.

   Summary of Findings:

   A slightly higher DNA yield was obtained when specimens were collected with the OMNIgene.GUT System than the fresh specimen, regardless of storage time (0 or 14 days at room temperature and frozen, respectively). While relative abundance of bacterial phyla detected were comparable among specimens collected with the OMNIgene.GUT System and those collected in a sterile container and either processed immediately or stored at -20C, significant differences were found at the genus level (P=0.003 and 0.004, respectively). Mean DNA yields were significantly higher when extraction was performed with the MP Kit than the MO BIO or QIA Kits (0.34 ± 0.018 ng/μl versus 0.09 ± 0.03 and 0.12 ± 0.02 ng/μl; respectively, P<0.001, both). The mean A260/A280 absorbance ratios were highest following extraction with the MP and QIA Kits (2.00 and 1.91, respectively) and the mean A260/A280 for the MO BIO Kit (1.55) was significantly lower (P<0.001, both). Overall, a higher average number of OTUs per specimen was detected with the MiSeq platform compared to the Ion Torrent platform (257.806 ± 23.076 versus 110.512 ± 7.529), with significant differences at both the family and genus levels (P=0.026 and P<0.001, respectively). Using the fresh immediately processed specimen as a reference point, the MO BIO Kit extracted a higher proportion of Bacteroidaceae (48.6%) compared to the MP and QIA extraction kits (33.4% and 24.8%, respectively), but the MP Kit was 1.9- to 3.2-fold more efficient at extracting all other families compared to the other two extraction kits (P<0.001, for all).

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • None (Fresh)
   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage conditions	Fresh Frozen OMNIgene.GUT at room temperature
   Storage	Time at room temperature	14 days
   Next generation sequencing Specific	Technology platform	Ilumina MiSeq Ion Torrent PGM platform
   Analyte Extraction and Purification	Analyte isolation method	MP Biomedicals Fast DNA SPIN Kit for Feces QIAgen QIAamp Fast DNA Stool Mini Kit MO BIO Power Fecal DNA Isolation Kit
   Storage	Storage duration	0 days 14 days

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