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Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Author(s): Hong BY, Driscoll M, Gratalo D, Jarvie T, Weinstock GM

Publication: Int J Mol Sci, 2024, Vol. 25, Page

PubMed ID: 38474213 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the microbiome sequencing results of fecal specimens after DNA was extracted by different methods (a novel alkaline-, heat-, and detergent-based method (Rapid) versus the PowerSoil Ki) and analyzed by different sequencers (Illumina 16S V1V3 and PacBio 16S V1V9). Additionally, the effect of extraction on observed microbial abundance was investigated following DNA extraction from a mock community (mix of 10 bacterial species) with 6 different methods (Rapid, Bead pasting, MasterPure Complete DNA and RNA Purification Kit, PowerSoil kit, QIAamp DNA Stool Kit and ZymoBIOMICSTM DNA/RNA Mini Kit). Feces were collected from ten hospitalized patients with lung cancer and ten “control” (diagnosis not specified) patients. Feces were stored at -80°C until DNA extraction. DNA was extracted from fecal specimens using the mechanical bead-beating-based Qiagen PowerSoil Kit and the rapid novel DNA Purification and 16S rRNA Amplification Kit that includes KOH, lysis buffer and heat (Rapid). Regions of V4 (292 bp) and V1V3 (526 bp) were PCR amplified and sequenced on an Illumina MiSeq instrument. A 1506 bp region of V1V9 was PCR amplified and sequenced on a Pacific Biosciences (PacBio) Sequel instrument. Illumina MiSeq reads were analyzed using the mothur phylotype pipeline while the PacBio reads were analyzed using the PacBio software, demultiplexed and classified using SBanalyzer with a custom algorithm. To clarify the impacts of extraction on observed microbial abundance, DNA was extracted from a “mock” community consisting of 10 bacterial species using the Rapid method, Bead pasting (a modification of the Bead Beater Lysis Kit), the MasterPure Complete DNA and RNA Purification Kit, the PowerSoil Kit, the QIAamp DNA Stool Kit and the ZymoBIOMICS DNA/RNA Mini Kit. The mock community DNA was sequenced as described for fecal specimens with the exception that data was analyzed at the species levels instead of the genus level.

   Summary of Findings:

   The observed relative abundance of bacterial species in the mock specimen was dependent on the extraction method used, with lower amounts of R. lactaris observed when extraction was with the PowerSoil Kit or QIAamp DNA Stool Kit and lower L. paracasei when extraction was with the MasterPure Complete DNA and RNA Purification Kit, PowerSoil kit, or the QIAamp DNA Stool Kit compared to the Rapid or Bead pasting methods. Importantly, the observed relative abundance did not match the true abundance of the mock community specimen for any of the extraction methods evaluated, and two species were under-represented or not observed following extraction with each of the six methods. The authors report that the observed relative abundance between technical replicates of the mock community was most consistent when extraction was with the Rapid method or Bead pasting.

   The authors established 1100 as the minimum number of reads necessary to accurately determine taxonomic diversity; however, after rarefaction, 2538 reads for Illumina V1V3 specimens from all 20 patients were included and 1057 reads for PacBio V1V9 specimens from 11 patients were included. Extraction of DNA from fecal specimens with the Rapid method, rather than the PowerSoil Kit, resulted in more taxa identified and increased diversity and evenness by PacBio 16S V1V9 amplicon sequencing (p=0.02, p=0.016 and p=0.02, respectively) and increased evenness by Illumina 16S V1V3 amplicon sequencing (p=0.013). Principal coordinate analysis (PCoA) based on ThetaYC distances (which considers relative proportions of species) showed significant differences between the two extraction methods by Illumina 16S V1V3 sequencing (p<0.001) and PacBio 16S V1V9 sequencing (p<0.006), but significant differences were not observed when PCoA was based on Jaccard distances (compares detection not abundance) using either sequencing method. The authors state that PcoA results indicate that the Rapid method lysed more cells and, based on diversity, it was a potential reason for the increased lysis of Firmicutes. Further analysis identified an increase in the abundance of Firmicutes and Actinobacteria species and a decrease in Bacteroidetes following extraction with the Rapid method compared to the PowerSoil Kit using either Illumina V1V3 or PacBio V1V9 sequencing. Non-metric multidimensional scaling (NMDS) plots confirmed that Bacteroidetes and Firmicutes were the drivers of the differences observed between the two extraction methods.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	PowerSoil Kit Rapid Method (alkaline, heat, and detergent based)
   Next generation sequencing Specific	Technology platform	Illumina 16S V1V3 sequencing Illumina 16S V4 sequencing PacBio 16S V1V9 sequencing
   Next generation sequencing Specific	Length of gene fragment	292 bp 526 bp 1506 bp

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