Reducing bias in microbiome research: Comparing methods from sample collection to sequencing
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Quality control
- Retracted paper
- Contamination issues suspected
- Batch effect issues suspected
- Uncontrolled confounding suspected
- Results are suspect (various reasons)
- Tags applied
Experiment 1
Subjects
- Location of subjects
- Netherlands
- Host species Species from which microbiome was sampled. Contact us to have more species added.
- Homo sapiens
- Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology
- Feces Cow dung,Cow pat,Droppings,Dung,Excrement,Excreta,Faeces,Fecal material,Fecal matter,Fewmet,Frass,Guano,Matières fécales@fr,Merde@fr,Ordure,Partie de la merde@fr,Piece of shit,Porción de mierda@es,Portion of dung,Portion of excrement,Portion of faeces,Portion of fecal material,Portion of fecal matter,Portion of feces,Portion of guano,Portion of scat,Portionem cacas,Scat,Spoor,Spraint,Stool,Teil der fäkalien@de,Feces,feces
- Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology
- Sample collection protocol Sample collection protocol,sample collection protocol
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Frozen Zymo control (-80-ZYCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature zymo buffer (RT-ZYBUF)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Zymo buffer at room temperature.
- Group 0 sample size Number of subjects in the control (unexposed) group
- 13
- Group 1 sample size Number of subjects in the case (exposed) group
- 13
- Antibiotics exclusion Number of days without antibiotics usage (if applicable) and other antibiotics-related criteria used to exclude participants (if any)
- 3 months
Lab analysis
- Sequencing type
- 16S
- 16S variable region One or more hypervariable region(s) of the bacterial 16S gene
- V4
- Sequencing platform Manufacturer and experimental platform used for quantifying microbial abundance
- Illumina
Statistical Analysis
- Data transformation Data transformation applied to microbial abundance measurements prior to differential abundance testing (if any).
- relative abundances
- Statistical test
- LEfSe
- Significance threshold p-value or FDR threshold used for differential abundance testing (if any)
- 0.05
- MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?
- Yes
- LDA Score above Threshold for the linear discriminant analysis (LDA) score for studies using the popular LEfSe tool
- 4
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary Fig 2a
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer In green, compared to the control samples in red.
Abundance in Group 1: increased abundance in Room temperature zymo buffer (RT-ZYBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidaceae | ||
| Bacteroidales | ||
| Bacteroides | ||
| Bacteroidia | ||
| Bacteroidota |
Revision editor(s): Shulamite
Signature 2
Source: Supplementary Fig 2a
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: decreased abundance in Room temperature zymo buffer (RT-ZYBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Blautia | ||
| Collinsella | ||
| Collinsella aerofaciens | ||
| Coriobacteriaceae | ||
| Coriobacteriales | ||
| Coriobacteriia |
Revision editor(s): Shulamite
Experiment 2
Subjects
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Zymo control at room temperature (RT-ZYCON)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Sample storage at room temperature without buffer
- Antibiotics exclusion Number of days without antibiotics usage (if applicable) and other antibiotics-related criteria used to exclude participants (if any)
- 3 Months
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary Fig 2b
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: increased abundance in Zymo control at room temperature (RT-ZYCON)
| NCBI | Quality Control | Links |
|---|---|---|
| Enterobacterales | ||
| Enterobacteriaceae | ||
| Gammaproteobacteria | ||
| Pseudomonadota | ||
| Escherichia/Shigella sp. |
Revision editor(s): Shulamite
Signature 2
Source: Supplementary Fig 2b
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: decreased abundance in Zymo control at room temperature (RT-ZYCON)
| NCBI | Quality Control | Links |
|---|---|---|
| Clostridia | ||
| Eubacteriales | ||
| Lachnospiraceae | ||
| Lachnospirales | ||
| Staphylococcus | ||
| Bacillota | ||
| Oscillospiraceae |
Revision editor(s): Shulamite
Experiment 3
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Zymo control at room temperature (RT-ZYCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Zymo buffer at room temperature (RT-ZYBUF).
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Zymo buffer at room temperature.
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary Fig 2c
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: increased abundance in Zymo buffer at room temperature (RT-ZYBUF).
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidaceae | ||
| Bacteroidales | ||
| Bacteroides | ||
| Bacteroidia | ||
| Bacteroidota | ||
| Clostridia | ||
| Faecalibacterium | ||
| Faecalibacterium prausnitzii | ||
| Bacillota |
Revision editor(s): Shulamite
Signature 2
Source: Supplementary Fig 2c
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: decreased abundance in Zymo buffer at room temperature (RT-ZYBUF).
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Collinsella | ||
| Collinsella aerofaciens | ||
| Coriobacteriaceae | ||
| Coriobacteriales | ||
| Coriobacteriia | ||
| Enterobacterales | ||
| Enterobacteriaceae | ||
| Escherichia/Shigella sp. |
Revision editor(s): Shulamite
Experiment 4
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Frozen OMNIgene GUT control (-80-OMCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature OMNIgene GUT buffer (RT-OMBUF)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- OMNIgene·GUT tube with a stabilization buffer and stored at RT for 3–5 days before freezing at −80°C
- Group 0 sample size Number of subjects in the control (unexposed) group
- 65
- Group 1 sample size Number of subjects in the case (exposed) group
- 65
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary Fig 2d
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: increased abundance in Room temperature OMNIgene GUT buffer (RT-OMBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidaceae | ||
| Bacteroidales | ||
| Bacteroides | ||
| Bacteroidia | ||
| Bacteroidota | ||
| Phocaeicola vulgatus |
Revision editor(s): Shulamite
Signature 2
Source: Supplementary Fig 2d
Description: LEfSe analysis showing the significantly distinguishing taxa between the different storage methods based on an LDA score >4.0. Showing the effect of storage at RT, with or without a stabilization buffer in green, compared to the control samples in red.
Abundance in Group 1: decreased abundance in Room temperature OMNIgene GUT buffer (RT-OMBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Bacillota | ||
| Bifidobacteriaceae | ||
| Bifidobacteriales | ||
| Bifidobacterium | ||
| Blautia | ||
| Clostridia | ||
| Coriobacteriales | ||
| Coriobacteriia | ||
| Lachnospiraceae | ||
| Lachnospirales | ||
| Acidimicrobiia |
Revision editor(s): Shulamite
Experiment 5
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Frozen zymo control (-80-ZYCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature zymo buffer (RT-ZYBUF)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Zymo buffer at room temperature.
- Group 0 sample size Number of subjects in the control (unexposed) group
- 13
- Group 1 sample size Number of subjects in the case (exposed) group
- 13
Lab analysis
Statistical Analysis
- Statistical test
- Mann-Whitney (Wilcoxon)
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Fig 3B
Description: Boxplots show the relative abundance of the five most abundant phyla. The difference between the storage conditions was tested with the Wilcoxon test.
Abundance in Group 1: increased abundance in Room temperature zymo buffer (RT-ZYBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidota | ||
| Pseudomonadota |
Revision editor(s): Shulamite
Signature 2
Source: Fig 3B
Description: Boxplots showing the relative abundance of the five top most abundant phyla.
Abundance in Group 1: decreased abundance in Room temperature zymo buffer (RT-ZYBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota |
Revision editor(s): Shulamite
Experiment 6
Subjects
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature zymo control (RT-ZYCON)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Room temperature zymo control
Lab analysis
Statistical Analysis
- LDA Score above Threshold for the linear discriminant analysis (LDA) score for studies using the popular LEfSe tool
- Not specified
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Fig 3B
Description: Boxplots show the relative abundance of the five top most abundant phyla.
Abundance in Group 1: increased abundance in Room temperature zymo control (RT-ZYCON)
| NCBI | Quality Control | Links |
|---|---|---|
| Pseudomonadota | ||
| Verrucomicrobiota |
Revision editor(s): Shulamite, KateRasheed
Signature 2
Source: Fig 3B
Description: Boxplots show the relative abundance of the five top most abundant phyla.
Abundance in Group 1: decreased abundance in Room temperature zymo control (RT-ZYCON)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacillota |
Revision editor(s): Shulamite
Experiment 7
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Frozen OMNIgene·GUT control (-80-OMCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature OMNIgene·GUTbuffer (RT-OMBUF)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- OMNIgene·GUT tube with a stabilization buffer and stored at RT for 3–5 days before freezing at −80°C
- Group 0 sample size Number of subjects in the control (unexposed) group
- 65
- Group 1 sample size Number of subjects in the case (exposed) group
- 65
Lab analysis
- Sequencing type
- WMS
- 16S variable region One or more hypervariable region(s) of the bacterial 16S gene
- Not specified
Statistical Analysis
- LDA Score above Threshold for the linear discriminant analysis (LDA) score for studies using the popular LEfSe tool
- 3
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Fig 3B
Description: Bacterial composition at the phylum level, showing the relative abundance of the phyla.
Abundance in Group 1: increased abundance in Room temperature OMNIgene·GUTbuffer (RT-OMBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidota | ||
| Pseudomonadota |
Revision editor(s): Shulamite
Signature 2
Source: Fig 3B
Description: Bacterial composition at the phylum level, showing the relative abundance of the phyla.
Abundance in Group 1: decreased abundance in Room temperature OMNIgene·GUTbuffer (RT-OMBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Bacillota |
Revision editor(s): Shulamite
Experiment 8
Subjects
- Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology
- Nucleic acid extraction protocol Nucleic acid extraction protocol,nucleic acid extraction protocol
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Mechanical disruption blood kit (MD-BK)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Enzymatical disruption blood kit (ED-BK)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Enzymatical disruption blood kit refers to enzymatical disruption (ED) that was purified with the Maxwell RSC Blood DNA kit (BK).
- Group 0 sample size Number of subjects in the control (unexposed) group
- 14
- Group 1 sample size Number of subjects in the case (exposed) group
- 14
Lab analysis
- Sequencing type
- 16S
- 16S variable region One or more hypervariable region(s) of the bacterial 16S gene
- V4
Statistical Analysis
- LDA Score above Threshold for the linear discriminant analysis (LDA) score for studies using the popular LEfSe tool
- Not specified
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Fig 8A
Description: Boxplots show the relative abundance of the top most abundant phyla. The Wilcoxon test was used to calculate the adjusted p-values of the differences between the DNA extraction methods.
Abundance in Group 1: increased abundance in Enzymatical disruption blood kit (ED-BK)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidota |
Revision editor(s): Shulamite
Signature 2
Source: Fig 8A
Description: Boxplots show the relative abundance of the top most abundant phyla. The Wilcoxon test was used to calculate the adjusted p-values of the differences between the DNA extraction methods.
Abundance in Group 1: decreased abundance in Enzymatical disruption blood kit (ED-BK)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Bacillota |
Revision editor(s): Shulamite
Experiment 9
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Mechanical disruption fecal kit (MD-FK)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Enzymatical disruption fecal kit (ED-FK)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Enzymatical disruption fecal kit refers to enzymatical disruption (ED) that was purified with the Maxwell RSC Fecal Microbiome DNA kit (FK).
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Fig 8A
Description: Boxplots show the relative abundance of the top most abundant phyla. The Wilcoxon test was used to calculate the adjusted p-values of the differences between the DNA extraction methods.
Abundance in Group 1: increased abundance in Enzymatical disruption fecal kit (ED-FK)
| NCBI | Quality Control | Links |
|---|---|---|
| Bacteroidota |
Revision editor(s): Shulamite
Signature 2
Source: Fig 8A
Description: Boxplots show the relative abundance of the top most abundant phyla. The Wilcoxon test was used to calculate the adjusted p-values of the differences between the DNA extraction methods.
Abundance in Group 1: decreased abundance in Enzymatical disruption fecal kit (ED-FK)
| NCBI | Quality Control | Links |
|---|---|---|
| Actinomycetota | ||
| Bacillota |
Revision editor(s): Shulamite
Experiment 10
Subjects
- Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology
- Sample collection protocol Sample collection protocol,sample collection protocol
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Frozen zymo control (-80-ZYCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature zymo control (RT-ZYCON)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Room temperature zymo control
- Group 0 sample size Number of subjects in the control (unexposed) group
- 13
- Group 1 sample size Number of subjects in the case (exposed) group
- 13
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary fig 3A
Description: Boxplots of the relative abundance of Enterobacteriaceae in all conditions.
Abundance in Group 1: increased abundance in Room temperature zymo control (RT-ZYCON)
| NCBI | Quality Control | Links |
|---|---|---|
| Enterobacteriaceae |
Revision editor(s): Shulamite, KateRasheed
Experiment 11
Subjects
- Group 0 name Corresponds to the control (unexposed) group for case-control studies
- Room temperature zymo control(RT-ZYCON)
- Group 1 name Corresponds to the case (exposed) group for case-control studies
- Room temperature zymo buffer (RT-ZYBUF)
- Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group
- Room temperature zymo buffer
Lab analysis
Statistical Analysis
Alpha Diversity
- Shannon Estimator of species richness and species evenness: more weight on species richness
- unchanged
- Simpson Estimator of species richness and species evenness: more weight on species evenness
- unchanged
- Richness Number of species
- unchanged
Signature 1
Source: Supplementary fig 3A
Description: Boxplots of the relative abundance of Enterobacteriaceae in all storage conditions.
Abundance in Group 1: decreased abundance in Room temperature zymo buffer (RT-ZYBUF)
| NCBI | Quality Control | Links |
|---|---|---|
| Enterobacteriaceae |
Revision editor(s): Shulamite, KateRasheed
