This study was undertaken with approval from the University of Adelaide Human Ethics Research Committee (H-2013-097 and H-78-2003). Parents provided written informed consent for the use of samples and the data for this research.
Our cohort of 221 Australian children consisted of 108 twin pairs, 2 unpaired twins and one set of triplets identified from the 550 twin/triplet families enrolled in the Tooth Emergence and Oral Health study. Parents/caregivers were required to complete a series of questionnaires as part of the study. The sex of participants was assigned based on parental report. Sex-specific effects were not detected from analysis of ARG diversity or gene abundance. Hence, sex was not included in further analysis of the resistome. A standard medical history was taken at the clinical examination at T3. See TableS5 , Supplementary Information for key population characteristics. At T3, the severity of caries was assessed using ICDAS II. In the 66 CA children, the mean ICDAS II score was 1 ± 1.8, while the remaining 145 were caries-free (ICDAS II score = 0).
From the 221 children, 542 oral biofilm samples were initially identified for genomic analysis, of which a total of 12 were excluded, making the final sample size 530. Two were excluded due to antibiotic use (within the past three months). Three were excluded due to extreme sequence depth variation from the mean. This included two with low sequence depth, T1657B_14042014_Q2_Q3 (0.339702 million target reads) and T1472B_25022007_D1_D2 (6.447160 million target reads), and one unusually high sequence depth sample, T1658A_6012009_D1_D2 (117.369 million target reads). We excluded seven samples that contained over 65% host DNA. The eligible samples were from 93 monozygous (MZ), 66 dizygous (DZ), and 59 opposite sex DZ (OSDZ) twins plus and one set of DZ/OSDZ/DZ triplets. One hundred and seventeen children (53%) were sampled at all three time points, 73 (35%) were sampled at 2 time points and 28 (12%) participants sampled at one time point only. While all twins/triplets were samples at the same time, not all individuals had a sample available that met the requirements for stage of dental development for all time points. For this reason, in addition to the post-sequencing removal of 12 samples, there was inconsistent sampling over time.
Our cohort of 221 Australian children consisted of 108 twin pairs, 2 unpaired twins and one set of triplets identified from the 550 twin/triplet families enrolled in the Tooth Emergence and Oral Health study. Parents/caregivers were required to complete a series of questionnaires as part of the study. The sex of participants was assigned based on parental report. Sex-specific effects were not detected from analysis of ARG diversity or gene abundance. Hence, sex was not included in further analysis of the resistome. A standard medical history was taken at the clinical examination at T3. See Table
From the 221 children, 542 oral biofilm samples were initially identified for genomic analysis, of which a total of 12 were excluded, making the final sample size 530. Two were excluded due to antibiotic use (within the past three months). Three were excluded due to extreme sequence depth variation from the mean. This included two with low sequence depth, T1657B_14042014_Q2_Q3 (0.339702 million target reads) and T1472B_25022007_D1_D2 (6.447160 million target reads), and one unusually high sequence depth sample, T1658A_6012009_D1_D2 (117.369 million target reads). We excluded seven samples that contained over 65% host DNA. The eligible samples were from 93 monozygous (MZ), 66 dizygous (DZ), and 59 opposite sex DZ (OSDZ) twins plus and one set of DZ/OSDZ/DZ triplets. One hundred and seventeen children (53%) were sampled at all three time points, 73 (35%) were sampled at 2 time points and 28 (12%) participants sampled at one time point only. While all twins/triplets were samples at the same time, not all individuals had a sample available that met the requirements for stage of dental development for all time points. For this reason, in addition to the post-sequencing removal of 12 samples, there was inconsistent sampling over time.
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