Tongue swab samples were collected from three healthy subjects at baseline, four weeks, and eight weeks from study initiation. DNA was extracted with the Qiagen DNeasy PowerSoil kit, and was prepared for shotgun metagenomic sequencing using either the Nextera XT kit (XT) or Nextera Flex DNA kit (Flex). Each kit was used with both the full reagent volume (1X), or with 1/4 reagent volume (1/4X). Libraries were sequenced on an Illumina HiSeq 2500 instrument.
Average nucleotide quality score after trimming adapter and filtering low quality (only forward read or reverse read or neither was kept after trimmomatic) read pairs is given. Red dots represent the mean quality score and vertical line segments represent mean \(\pm\) sample standard deviation.
The dashed line represents 1 million read counts.
Here, we focus on Streptococcus, the predominant genus.
##
## Call:
## lm(formula = log10(Proportion) ~ Specimen + lib_prep + rxn_scale,
## data = .)
##
## Residuals:
## Min 1Q Median 3Q Max
## -0.030765 -0.007870 0.001439 0.006046 0.025389
##
## Coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) -0.820360 0.008081 -101.517 < 2e-16 ***
## SpecimenSpecimen 1064 -0.442511 0.010130 -43.683 < 2e-16 ***
## SpecimenSpecimen 1897 -0.654976 0.010130 -64.657 < 2e-16 ***
## SpecimenSpecimen 2005 -0.444399 0.011013 -40.354 < 2e-16 ***
## SpecimenSpecimen 2132 -0.238782 0.010130 -23.572 < 2e-16 ***
## SpecimenSpecimen 2134 -0.377983 0.010130 -37.313 < 2e-16 ***
## SpecimenSpecimen 2151 0.142903 0.010130 14.107 3.49e-12 ***
## SpecimenSpecimen 820 -0.620535 0.010130 -61.257 < 2e-16 ***
## lib_prepXT -0.027622 0.005179 -5.334 2.74e-05 ***
## rxn_scale1X 0.001416 0.005179 0.273 0.787
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 0.01433 on 21 degrees of freedom
## Multiple R-squared: 0.9981, Adjusted R-squared: 0.9972
## F-statistic: 1208 on 9 and 21 DF, p-value: < 2.2e-16
## Analysis of Variance Table
##
## Response: log10(Proportion)
## Df Sum Sq Mean Sq F value Pr(>F) R-squared
## Specimen 7 2.22524 0.31789 1548.9013 0.00000 0.99544
## lib_prep 1 0.00588 0.00588 28.6275 0.00003 0.00263
## rxn_scale 1 0.00002 0.00002 0.0747 0.78724 0.00001
## Residuals 21 0.00431 0.00021 0.00193
Alpha diversity (within sample diversity) was assessd by the Shannon index.
##
## Call:
## lm(formula = ShannonIdx ~ Specimen + lib_prep + rxn_scale, data = alpha_df)
##
## Residuals:
## Min 1Q Median 3Q Max
## -0.12329 -0.05014 -0.01370 0.03130 0.21555
##
## Coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) 3.101521 0.050256 61.714 < 2e-16 ***
## SpecimenSpecimen 1064 -0.202864 0.063000 -3.220 0.00411 **
## SpecimenSpecimen 1897 -0.110428 0.063000 -1.753 0.09422 .
## SpecimenSpecimen 2005 -0.193912 0.068488 -2.831 0.01000 *
## SpecimenSpecimen 2132 -0.096956 0.063000 -1.539 0.13874
## SpecimenSpecimen 2134 -0.035943 0.063000 -0.571 0.57437
## SpecimenSpecimen 2151 -0.163412 0.063000 -2.594 0.01694 *
## SpecimenSpecimen 820 -0.088616 0.063000 -1.407 0.17417
## lib_prepXT 0.005965 0.032208 0.185 0.85484
## rxn_scale1X 0.060327 0.032208 1.873 0.07505 .
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 0.08909 on 21 degrees of freedom
## Multiple R-squared: 0.4936, Adjusted R-squared: 0.2765
## F-statistic: 2.274 on 9 and 21 DF, p-value: 0.05814
Beta diversity (similarity between samples) was assessed by Bray-Curtis distance.
##
## Call:
## adonis(formula = d ~ Specimen + lib_prep + rxn_scale, data = s)
##
## Permutation: free
## Number of permutations: 999
##
## Terms added sequentially (first to last)
##
## Df SumsOfSqs MeanSqs F.Model R2 Pr(>F)
## Specimen 7 2.51299 0.35900 241.664 0.98140 0.001 ***
## lib_prep 1 0.01368 0.01368 9.207 0.00534 0.001 ***
## rxn_scale 1 0.00276 0.00276 1.859 0.00108 0.160
## Residuals 21 0.03120 0.00149 0.01218
## Total 30 2.56062 1.00000
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1