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Methodology
Damien Roux
Intensive Care Unit, Louis Mourier Hospital, 178 rue des renouillers 92700 Colombes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6103-6416
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Characterization of the respiratory tract bacterial microbiota is in its infancy when compared to the gut microbiota knowledge. As key methodological steps can directly affect the accuracy of the results, it is crucial to determine a robust methodology in order to limit bias.
Two different pairs of primers 515F-806R targeting the V4 hypervariable region of the 16SrRNA gene, “S-V4” for “Stringent V4” primer pair and “R-V4” for “Relaxed V4” primer pair, are respectively used in two ongoing international projects “the Human microbiome project” and “the Earth microbiome project”.
We compared two methods of sample processing using these two different primer pairs for bacterial microbiota analyses of respiratory samples from critically-ill ventilated patients. For the later, Illumina 250 paired-end sequencing was done on a MiSeq platform after DNA extraction using mechanical lysis (bead-beating) and NucliSENS easyMAG. The concordance with conventional microbiology is the criterion of choice to determine the optimal method.
Results
Twenty samples from seven patients and four controls were sequenced. The two primer pair provided highly different results. Only 54% of the samples had a similar microbial composition with both pairs of primers. “S-V4” gave the best agreement with the conventional microbiology for ETA: 89% as compared to 44% for the “R-V4” primer pair. The main difference related to Enterobacteriaceae, which were concordant between conventional cultures and microbiota analyses using “S-V4”. Enterobacteriaceae detection was poor for “R-V4”. Among patients with VAP, a decrease in alpha diversity in ETA was observed. The mean of pairwise Unifrac distance was higher inside this group of patients at the time of VAP diagnosis as compared to control patients.
Conclusion
Accuracy of the bacterial lung microbiota composition was highly correlated to the pair of primers used for amplification of the 16s rRNA hypervariable sequence. Comparison of microbiota results obtained by sequencing and conventional microbiology allowed us to select the best option for further studies. This work validates our methodology based on 16SrRNA gene amplification with 515F-806R “S-V4” primer pair associated to Illumina® MiSeq 250 paired-end sequencing.
Keywords
Lung microbiome, 16S rRNA gene, high-throughput sequencing, dysbiosis, metagenomics, ventilator-associated pneumonia, mechanical ventilation
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This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.docx
Additional file 1. Per sequence quality measured by Phred score for all samples and all V4 primer pairs The first histogram represents for a total of 168 samples the number of reads with average quality score. Samples amplified with “V4 Human primer pair” and “V4 Earth primer pair” are pooled together. Samples passed the filter if mean Phred score was > 27. Samples passed the filter if 20 < Phred score < 27. Samples failed if Phred score was <20. Globally per sequence quality was excellent with 152 samples passing the filter, 4 samples passing the filter with warning ad 12 failed. The second histogram represents the mean Phred score value across each base position for all the 168 samples. Quality was still excellent with 109 samples passing the filter, 22 passing the filterwith warning and 37 failed.
- Additionalfile2.xlsx
Additional file 2. Global table count of identified OTU for both V4 primer pairs For each sample, all OTU identified at the family level are mentioned the fifth column in descending order, taking into account the number of reads per OTU. For each OTU identified number of reads are notified in the sixth column.
- Additionalfile3.docx
Additional file 3. Subject enrollment and antibiotic exposure Endotracheal aspiration of seven intubated patient were sequenced in this study. All except one patient had a CAP or aspiration pneumonia on admission so received antibiotics since D0. All patients received Amoxicillin, Amoxicilline-clavulanate acid or Cefotaxime for CAP. All VAP patient presented late onset VAP as the day of diagnosis was after D7. NA indicates that the patient received no antibiotic.
- Additionalfile4.pdf
Additional file 4. Alpha diversity estimates for each V4 primer pair Comparison between Shannon index (Figure S4A) and number of OTUs obtained with the rarefaction threshold of 5.103 for all samples and controls with each primer pair represented by a dotplot (Figure S4B). Samples collected on D-before for both VAP patients and control patients are represented in green. Samples collected on D-VAP are represented in blue. Positive control is represented in red. Samples from lower respiratory tract are represented with a triangle and these from upper respiratory tract are represented with a square.
- Additionalfile5.xlsx
Additional file 5. Comparison table of OTUs identified with each pair of primer For each samples the three most abundant OTU identified with “S-V4”primer pair and “R-V4” primer pair are represented. Only OTUS whose relative abundance represents more than 1% of the total are represented. The agreement between the two primer pairs is retained if the two most abundant OTUs are the same. OTUs are represented at the genus level and classified in order of relative abundance for each sample.
- Additionalfile6.tif
Additional file 6. Pairwise Bray Curtis distance for “S-V4” primer pair across respiratory tract site and time Bray Curtis distance between all URT samples (left) and for all LRT samples (right) within each group : control patient at D0, VAP patients at D-Before and VAP patients at D-VAP.
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Methodology
Damien Roux
Intensive Care Unit, Louis Mourier Hospital, 178 rue des renouillers 92700 Colombes, France
Corresponding Author
ORCiD: https://orcid.org/0000-0002-6103-6416
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 04 Nov, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Characterization of the respiratory tract bacterial microbiota is in its infancy when compared to the gut microbiota knowledge. As key methodological steps can directly affect the accuracy of the results, it is crucial to determine a robust methodology in order to limit bias.
Two different pairs of primers 515F-806R targeting the V4 hypervariable region of the 16SrRNA gene, “S-V4” for “Stringent V4” primer pair and “R-V4” for “Relaxed V4” primer pair, are respectively used in two ongoing international projects “the Human microbiome project” and “the Earth microbiome project”.
We compared two methods of sample processing using these two different primer pairs for bacterial microbiota analyses of respiratory samples from critically-ill ventilated patients. For the later, Illumina 250 paired-end sequencing was done on a MiSeq platform after DNA extraction using mechanical lysis (bead-beating) and NucliSENS easyMAG. The concordance with conventional microbiology is the criterion of choice to determine the optimal method.
Results
Twenty samples from seven patients and four controls were sequenced. The two primer pair provided highly different results. Only 54% of the samples had a similar microbial composition with both pairs of primers. “S-V4” gave the best agreement with the conventional microbiology for ETA: 89% as compared to 44% for the “R-V4” primer pair. The main difference related to Enterobacteriaceae, which were concordant between conventional cultures and microbiota analyses using “S-V4”. Enterobacteriaceae detection was poor for “R-V4”. Among patients with VAP, a decrease in alpha diversity in ETA was observed. The mean of pairwise Unifrac distance was higher inside this group of patients at the time of VAP diagnosis as compared to control patients.
Conclusion
Accuracy of the bacterial lung microbiota composition was highly correlated to the pair of primers used for amplification of the 16s rRNA hypervariable sequence. Comparison of microbiota results obtained by sequencing and conventional microbiology allowed us to select the best option for further studies. This work validates our methodology based on 16SrRNA gene amplification with 515F-806R “S-V4” primer pair associated to Illumina® MiSeq 250 paired-end sequencing.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
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