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Table 2 Extraction and sequencing controls included in this study

From: Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens

Control type

Control subtype

Control name

Composition

Source

DNA extraction performed in our laboratory

DNA extraction kit

Extraction replicates

Sequencing replicates

Total included for equencing

Extraction controls

Bacterial mock communities

Zymobiomics-Primestore-high

900 μl of Zymobiomics-Cells suspended in 3600 μl Primestore

Zymo Research Corp., Irvine, CA, United States

&

Longhorn Vaccines & Diagnostics, Bethesda, MD, USA

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

  

Zymobiomics-STGG-high

900 μl of Zymobiomics-Cells suspended in 3600 μl STGG

Zymo Research Corp., Irvine, CA, United States & National Health Laboratory Services, Cape Town, South Africa

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

  

Zymobiomics-Primestore-low

1-in-104 fold dilution of Zymobiomics-Primestore-high

Zymo Research Corp., Irvine, CA, United States & Longhorn Vaccines & Diagnostics, Bethesda, MD, USA

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

  

Zymobiomics-STGG-low

1-in-104 fold dilution of Zymobiomics-STGG-high

Zymo Research Corp., Irvine, CA, United States & National Health Laboratory Services, Cape Town, South Africa

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

 

NTCs

Primestore

Storage buffer Primestore

Longhorn Vaccines & Diagnostics, Bethesda, MD, USA

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

  

STGG

Storage buffer STGG

National Health Laboratory Services, Cape Town, South Africa

Kit-QS

Kit-ZB

3 per DNA extraction kit

6

Sequencing controls

Bacterial mock community DNA

BEI-DNA

1-in-10 fold dilution of HM-783D in Milli-Q® ultrapure water

BEI Resources, NIAID, NIH as part of the Human Microbiome Project, Manassas, VA, USA & MilliporeSigma, Burlington, MA, USA

x

Not specified

3

  

Zymobiomics-DNA

1-in-10 fold dilution of ZymoBIOMICS™ Microbial Community DNA Standard in Milli-Q® ultrapure water

Zymo Research Corp., Irvine, CA, United States & MilliporeSigma, Burlington, MA, USA

x

Not specified

8

 

Technical repeats

Within-run repeats

NP and IS specimens stored in Primestore

Collected from infants enrolled in the DCHS [26]

Kit-QS

2 per specimen

86

  

Between-run repeats

NP and IS specimens stored in Primestore

Collected from infants enrolled in the DCHS [26]

Kit-QS

2, 3 or 4 per specimen

123

 

NTCs

Primestore

Storage buffer Primestore

Longhorn Vaccines & Diagnostics, Bethesda, MD, USA

Kit-QS

35

  1. Zymobiomics-Cells ZymoBIOMICS™ Microbial Community Standard bacterial cells (Catalog No. D6300, Zymo Research Corp., Irvine, CA, United States), Kit-QS DSP Virus/Pathogen Mini Kit® using QIAsymphony® SP instrument (catalogue no. 937036, Qiagen GmbH, Hilden, Germany), Kit-ZB ZymoBIOMICS DNA Miniprep Kit (catalogue no. ZR D4300, Zymo Research Corp., Irvine, CA, United States), NTCs No template controls, Primestore PrimeStore® Molecular Transport medium (Longhorn Vaccines & Diagnostics Bethesda, MD, USA), STGG transport medium containing Skim-milk, Tryptone, Glucose and Glycerol, NP Nasopharyngeal swabs IS Induced sputum, DCHS Drakenstein Child Health Study