Reducing bias in microbiome research: Comparing methods from sample collection to sequencing.
Author(s): Kool J, Tymchenko L, Shetty SA, Fuentes S
Publication: Front Microbiol, 2023, Vol. 14, Page 1094800
PubMed ID: 37065158 PubMed Review Paper? No
Purpose of Paper
This paper compared the microbiome of case-matched fecal specimens that were immediately frozen with those that were stored for 3-5 days at room temperature either without preservation, in Zymo tubes (containing DNA/RNA shield), or in OMNIgene GUT tubes (containing stabilization buffer) by next-generation sequencing (NGS). Potential effects of lysis method, DNA extraction method, DNA input amount, the number of PCR cycles, DNA extraction in one versus multiple instrument runs, and using one set of barcodes or multiple sets on the fecal microbiome were also examined.
Conclusion of Paper
Although the Shannon index, Observed taxa, and Simpson’s indices were not affected by the use of stabilization solutions during fecal storage, nor did they differ between collection cohorts, clustering based on principal coordinate analysis (PcoA) was driven by cohort and to a lesser extent use of OMNIgene GUT tubes. Consequently, additional comparisons were limited to matched specimens within individual cohorts. Differences in the relative abundance of phyla and taxa were observed between frozen specimens and case-matched specimens that were stored for 3-5 days without buffer, in Zymo, or in OMNIgene GUT tubes.
For fecal specimens that were frozen without buffer, DNA yields (total and bacterial) were higher using both kits when lysis was with beads rather than proteinase K buffer, but there was no clear difference between the two extraction kits. In contrast, for fecal specimens frozen in Zymo buffer, DNA yields (total and bacterial) were higher when DNA was extracted using the Maxwell RSC Fecal Microbiome DNA Kit than the Maxwell RSC Blood DNA Kit, but no clear effect of lysis method was observed. Shannon index, Observed taxa, and Simpson’s indices were unaffected by DNA extraction kit or lysis method, but PcoA based on the Bray–Curtis distance showed an effect of lysis method. Differences at the phyla level were noted between lysis methods for each extraction kit, but not between extraction kits for specimens lysed using either method. Using mock community samples (commercially available combinations of bacterial strains) the correlation with the known composition was the strongest when mechanical disruption was combined with the Maxwell RSC Fecal Microbiome DNA Kit. DNA input and the number of PCR cycles did not affect the Shannon index, Observed taxa, Simpson’s indices, or Bray–Curtis distances, but a higher number of PCR cycles resulted in the detection of microbial reads in negative controls. Divergence in Bray-Curtis distances were smaller when the same bar-codes were used than when different barcodes were used and when extraction occurred in one run versus different runs, but the differences were only significant when a mix of five specimens was used, not mock communities.
Studies
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Study Purpose
This study compared the microbiome of case-matched fecal specimens that were immediately frozen with those that were stored for 3-5 days at room temperature either without preservation, in Zymo tubes (containing DNA/RNA shield), or in OMNIgene GUT tubes (containing stabilization buffer) by next-generation sequencing (NGS). Fecal specimens from 12 healthy volunteers were aliquoted and matched specimens were frozen immediately at -80°C or stored at room temperature for 3–5 days without buffer or in Zymo tubes before freezing. Fecal specimens from 64 participants in the PIENTER study (diagnosis not specified) were aliquoted and matched specimens were frozen immediately at -80°C or collected in OMNIgene GUT tubes and stored at room temperature for 3–5 days before freezing. DNA was extracted by lysis (unclear if it was mechanical or enzymatic) followed by purification using the Maxwell RSC Fecal Kit. DNA was quantified by fluorometer and by real-time PCR amplification of the 16S rRNA gene. Libraries were constructed by V4 amplicon PCR and sequenced on a MISeq Instrument.
Summary of Findings:
Although Shannon index, Observed taxa, and Simpson’s indices were not affected by the use of stabilization solutions during fecal storage (OMNIgene or Zymo), nor did they differ between collection cohorts clustering based on PcoA was driven by cohort (R2=0.026; P=0.001) and to a lesser extent use of OMNIgene GUT tubes (R2=0.020; P=0.002). Consequently, additional comparisons were limited to matched specimens within individual cohorts. Compared to specimens frozen immediately, those that were stored for 3-5 days at room temperature without buffer had fewer firmicutes (P=0.000488) and more proteobacteria (P=0.000488) and verrucomicribiota (P=0.0143), and specimens stored for 3-5 days at room temperature in Zymo tubes had more bacteroidota (P=0.000488) and proteobacteria (P=0.00342) and fewer actinobacteriota (P=0.000488). Compared to specimens frozen immediately, those stored for 3-5 days at room temperature in OMNIgene GUT tubes had fewer firmicutes (P=0.0025) and actinobacteriota (P=7.87e-5) and more bacteroidota (P=2.32e-9), and proteobacteria (P= 2.91e-6). At the taxa level, there were differences between frozen specimens and specimens stored for 3-5 days without preservation or in Zymo or OMNIgene GUT tubes, but the differences were specific to the tube type. More taxa were differently expressed between frozen specimens and matched specimens stored at room temperature in OMNIgene GUT tubes than between matched frozen specimens and those stored at room temperature without buffer or in Zymo tubes.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
Diagnoses:
- Not specified
- Normal
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 0 days
3-5 days
Biospecimen Preservation Type of fixation/preservation Frozen
Zymo DNA/RNA Shield
OMNIgene GUT
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Study Purpose
This study compared the microbiome (by NGS) and DNA yield of matched fecal specimens that were immediately frozen with those that were stored for 3-5 days at room temperature in Zymo tubes (containing DNA/RNA shield) prior to lysis by either mechanical disruption or enzymatic lysis and extraction with either the Maxwell RSC Fecal Kit or Maxwell RSC Blood DNA Kit. Potential effects associated with DNA input amount, the number of PCR cycles, extraction in a single run versus multiple runs, and using one set of barcodes rather than multiple sets were also examined. Fecal specimens from 5 volunteers were divided and frozen immediately or stored at room temperature for 3-5 days in Zymo tubes containing DNA/RNA shield. DNA was extracted using either mechanical disruption (bead beating with 0.5 g zirconia/silica beads (0.1 mm) and five glass beads (2.7 mm) in STAR buffer or stabilization buffer) or by enzymatic lysis in a buffer containing Proteinase K followed by purification using the Maxwell RSC Fecal Kit or the Maxwell RSC Blood DNA Kit. PCR inhibitors were removed with the OneStep PCR Inhibitor Removal Kit. Total DNA was quantified by fluorometer and bacterial DNA yield was determined by real-time PCR amplification of the 16S rRNA gene. Libraries were constructed by V4 amplicon PCR and sequenced on a MISeq Instrument. To investigate the effect of DNA input and PCR cycles on the observed microbiome, sequencing libraries were constructed from DNA isolated from three fecal specimens using different DNA input amounts (16, 125, and 1,000 pg) and numbers of PCR cycles (25, 30, or 35). To investigate sources of variation, DNA was extracted from 20 mock communities (commercially available combinations of bacterial strains) and 23 specimens constructed by mixing 5 fecal specimens in multiple runs or in a single run, and sequencing within replicates with the same barcodes or different bar codes.
Summary of Findings:
For specimens frozen without buffer, DNA yields (total and bacterial) were higher using either kit when lysis was with beads rather than Proteinase K buffer, but there was no clear difference between the two extraction kits. In contrast, for specimens frozen in Zymo buffer, DNA yields (total and bacterial) were higher when DNA was extracted using the Maxwell RSC Fecal Microbiome DNA Kit than the Maxwell RSC Blood DNA Kit, but no clear effect of lysis method was observed. Shannon index, Observed taxa and Simpson’s indices were unaffected by the extraction kit or lysis method, but PCoA based on the Bray–Curtis distance showed an effect of lysis method (R2 =0.08585; p-adjusted=0.001). Mechanically lysed specimens had fewer firmicuites and actinobacteriota and more bacteriodota than enzymatically lysed specimens extracted using the same kit (P<0.05, all), but no differences at the phyla level were noted between the extraction kits for specimens lysed using either method. Using mock community samples (commercially available combinations of bacterial strains) the correlation with the known composition was strongest when mechanical disruption was combined with the Maxwell RSC Fecal Microbiome DNA kit (ρ=0.933). DNA input and the number of PCR cycles did not affect the Shannon index, Observed taxa, Simpson’s indices or Bray–Curtis distances, but the higher number of PCR cycles resulted in the detection of microbial reads in negative controls. Usinsg the mock community samples the correlation with the known composition was strongest when libraries were constructed using 125 pg input DNA and 25 PCR cycles (ρ=0.833). Divergence in Bray-Curtis distances were smaller when the same bar-codes were used than when different barcodes were used and when DNA was extracted in one run versus different runs, but the differences were only significant when a mix of five specimens was used, not mock communities.
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Maxwell RSC Fecal Microbiome DNA kit
Maxwell RSC Blood DNA kit
Single run
Multiple runs
Analyte Extraction and Purification Cell/tissue permeabilization Enzymatic lysis
Mechanical lysis
Next generation sequencing Specific Template/input amount 16 pg
125 pg
1,000 pg
Next generation sequencing Specific Nucleic acid amplification 25 cycles
30 cycles
35 cycles
Next generation sequencing Specific Template modification Replicates with different barcodes
Replicates with same barcodes