Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens
| dc.contributor.author | Claassen-Weitz, Shantelle | |
| dc.contributor.author | Gardner-Lubbe, Sugnet | |
| dc.contributor.author | Mwaikono, Kilaza S | |
| dc.contributor.author | du Toit, Elloise | |
| dc.contributor.author | Zar, Heather J | |
| dc.contributor.author | Nicol, Mark P | |
| dc.date.accessioned | 2020-05-18T13:46:22Z | |
| dc.date.available | 2020-05-18T13:46:22Z | |
| dc.date.issued | 2020-05-12 | |
| dc.date.updated | 2020-05-18T11:20:31Z | |
| dc.description.abstract | Careful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens. Results We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination. Conclusions We provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results. | |
| dc.identifier.apacitation | Claassen-Weitz, S., Gardner-Lubbe, S., Mwaikono, K. S., du Toit, E., Zar, H. J., & Nicol, M. P. (2020). Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens. <i>BMC Microbiology</i>, 20(1), 113. | en_ZA |
| dc.identifier.chicagocitation | Claassen-Weitz, Shantelle, Sugnet Gardner-Lubbe, Kilaza S Mwaikono, Elloise du Toit, Heather J Zar, and Mark P Nicol "Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens." <i>BMC Microbiology</i> 20, 1. (2020): 113. | en_ZA |
| dc.identifier.citation | Claassen-Weitz, S., Gardner-Lubbe, S., Mwaikono, K.S., du Toit, E., Zar, H.J. & Nicol, M.P. 2020. Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens. <i>BMC Microbiology.</i> 20(1):113. | en_ZA |
| dc.identifier.ris | TY - Journal Article AU - Claassen-Weitz, Shantelle AU - Gardner-Lubbe, Sugnet AU - Mwaikono, Kilaza S AU - du Toit, Elloise AU - Zar, Heather J AU - Nicol, Mark P AB - Careful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens. Results We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination. Conclusions We provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results. DA - 2020-05-12 DB - OpenUCT DP - University of Cape Town IS - 1 J1 - BMC Microbiology KW - 16S rRNA gene KW - Bacteriome KW - Contamination KW - High-throughput sequencing KW - Low biomass KW - Mock controls KW - Negative controls KW - Optimization KW - Reproducibility KW - Respiratory LK - https://open.uct.ac.za PY - 2020 T1 - Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens TI - Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens UR - ER - | en_ZA |
| dc.identifier.uri | https://doi.org/10.1186/s12866-020-01795-7 | |
| dc.identifier.uri | https://hdl.handle.net/11427/31901 | |
| dc.identifier.vancouvercitation | Claassen-Weitz S, Gardner-Lubbe S, Mwaikono KS, du Toit E, Zar HJ, Nicol MP. Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens. BMC Microbiology. 2020;20(1):113. . | en_ZA |
| dc.language.rfc3066 | en | |
| dc.rights.holder | The Author(s) | |
| dc.source | BMC Microbiology | |
| dc.source.journalissue | 1 | |
| dc.source.journalvolume | 20 | |
| dc.source.pagination | 113 | |
| dc.source.uri | https://bmcmicrobiol.biomedcentral.com/ | |
| dc.subject | 16S rRNA gene | |
| dc.subject | Bacteriome | |
| dc.subject | Contamination | |
| dc.subject | High-throughput sequencing | |
| dc.subject | Low biomass | |
| dc.subject | Mock controls | |
| dc.subject | Negative controls | |
| dc.subject | Optimization | |
| dc.subject | Reproducibility | |
| dc.subject | Respiratory | |
| dc.title | Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens | |
| dc.type | Journal Article |