Observed # of species (normalized) Chao 1 (richness) Nonsmoker 21 Male 2418 (337) 67 (33)
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1 Additional Files The human laryngeal microbiome: effects of cigarette smoke and reflux Marie E. Jetté, Kimberly A. Dill-McFarland, Alissa S. Hanshew, Garret Suen, Susan L. Thibeault Table S1. Detailed Participant Characteristics Cohort Age Gender # of sequences (normalized*) Observed # of species (normalized) Chao 1 (richness) Good s coverage 1/Simpson (diversity) Shannon (diversity) Nonsmoker 21 Male 2418 (337) 67 (33) GERD 22 Female 737 (321) 91 (51) Male 578 (350) 66 (66) Female 2555 (333) 89 (49) Female 5595 (333) 109 (50) Female 1002 (326) 128 (70) Male 1003 (329) 101 (61) Female 775 (325) 98 (62) Male 1502 (332) 81 (50) Female 5623(329) 136 (41) Male 1008 (327) 121 (63) Male (334) 134 (15) Female 709 (328) 74 (48) Female 495 (345) 71 (71) Male 506 (346) 55 (55) Male 752 (331) 62 (41) LPR 59 Female 709 (320) 82 (42) Female 509 (348) 43 (43) Female 1225 (341) 73 (53) Female 2525 (333) 71 (44) Male 2222 (329) 111 (54) Male 2180 (334) 100 (61) Female 1530 (330) 105 (58) Male 1941 (341) 87 (56) Male 1093 (340) 95 (63) Male 1756 (340) 74 (46) Male 347 (344) 38 (38) Male (334) 145 (47) Female 540 (346) 52 (52) Female 1548 (335) 84 (47) Female 641 (354) 57 (57) Female (327) 154 (39) Female (325) 154 (35) Female 462 (343) 37 (37) Female 755 (328) 67 (42) Male 2214 (337) 70 (49)
2 Normal 33 Male (335) 135 (34) Female (330) 135 (22) Female 4817 (327) 115 (40) Male 775 (332) 66 (45) Female 911 (329) 77 (48) Female 949 (334) 79 (50) Male 7926 (334) 96 (41) Female (330) 141 (37) Male 7421 (334) 139 (55) Female 1141 (335) 76 (57) Male 754 (329) 68 (43) Female 3952 (332) 104 (40) Female 1810 (336) 84 (45) Female 5856 (333) 104 (33) Female 1556 (330) 86 (54) Female 2121 (334) 70 (42) Male 7509 (328) 128 (45) Male 808 (336) 60 (44) Male 749 (321) 77 (40) Female 856 (327) 75 (38) Female 1131 (342) 66 (47) Male 746 (326) 73 (41) Female 396 (345) 55 (55) Male 720 (328) 61 (31) Female 860 (333) 67 (42) Female 5340 (338) 106 (42) Female 3050 (331) 95 (37) Female 780 (334) 43 (28) Female 1624 (338) 64 (41) Male 1404 (331) 88 (48) Male 922 (336) 63 (48) Female (338) 133 (40) Male 1621 (331) 69 (41) Female 747 (336) 60 (35) Male 4879 (334) 99 (31) Male 415 (344) 57 (57) Female 5249 (331) 93 (42) Male 3494 (334) 93 (47) Male 531 (345) 51 (51) Male 3148 (332) 88 (36) Female 484 (344) 58 (58) Cohort Age Gender # of sequences (normalized) Observed # of species (normalized) Chao 1 (richness) Good s coverage 1/Simpson (diversity) Shannon (diversity) Smoker GERD 25 Male 1205 (339) 79 (59)
3 LPR Normal 26 Male 341 (341) 46 (46) Female 1202 (335) 114 (69) Male 8580 (334) 120 (46) Female 3167 (336) 93 (34) Female 2741 (328) 67 (20) Female 861 (329) 52 (30) Female 2558 (335) 145 (72) Male 949 (330) 79 (51) Female 1366(329) 41 (23) Female 1760 (338) 63 (39) Male 2540 (334) 78 (36) Male 5514 (332) 133 (57) Female 4393 (336) 70 (23) Female 2202 (336) 70 (39) Male 1799 (331) 96 (42) Male 1474 (335) 46 (27) Male 1495 (329) 101 (51) Female 917 (337) 29 (17) Female 716 (338) 23 (20) *Following calculation of Good s coverage, all sequences were normalized to 340 sequences using the following calculation: (n sequences of OTU in x sample/total n sequences in x sample) * 340. Due to rounding to a whole number, the summed number of normalized sequences in each sample is variable. All downstream analyses were performed on normalized data.
4 Table S2. OTU Counts and Classifications (see.xlsx file)
5 Table S3. ANOVA and Tukey Testing of Diversity Metrics ANOVA chao shannon invsimpson smoke reflux age sex random smoke*reflux TukeyHSD smoker vs nonsmoker LPR vs GERD Norm vs GERD Norm vs LRP TukeyHSD Smoker:Normal vs NonSmoker:GERD Smoker:Normal vs NonSmoker:LPR Smoker:Normal vs NonSmoker:Normal Smoker:GERD vs NonSmoker:GERD NonSmoker:LPR vs NonSmoker:GERD Smoker:LPR vs NonSmoker:GERD NonSmoker:Normal vs NonSmoker:GERD NonSmoker:LPR vs Smoker:GERD Smoker:LPR vs Smoker:GERD NonSmoker:Normal vs Smoker:GERD Smoker:Normal vs Smoker:GERD Smoker:LPR vs NonSmoker:LPR NonSmoker:Normal vs NonSmoker:LPR NonSmoker:Normal vs Smoker:LPR Smoker:Normal vs Smoker:LPR
6 Table S4. PERMANOVA Analyses (p-values) of Bray-Curtis and Jaccard Indices BRAY-CURTIS JACCARD SMOKE REFLUX AGE SEX RANDOM SMOKE*REFLUX
7 Table S5. SIMPER Analysis of PERMANOVA SMOKE %* PHYLUM CLASS ORDER FAMILY GENUS OTU Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus OTU Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae unclassified OTU Bacteroidetes Flavobacteriia Flavobacteriales Weeksellaceae Cloacibacterium OTU Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae Helicobacter OTU Bacteroidetes Bacteroidia Bacteroidales Prevotellaceae Prevotella OTU Firmicutes Bacilli Lactobacillales Streptococcaceae Streptococcus OTU Actinobacteria Actinobacteria Actinomycetales Propionibacteriaceae Propionibacterium OTU Firmicutes Bacilli Bacillales Paenibacillaceae Paenibacillus OTU Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae unclassified OTU Firmicutes Clostridia Clostridiales Veillonellaceae unclassified OTU Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Moraxella OTU Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus OTU Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Acinetobacter OTU Firmicutes Bacilli Bacillales unclassified unclassified OTU Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas OTU Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas OTU Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Aquabacterium OTU Bacteroidetes Bacteroidia Bacteroidales Paraprevotellaceae Prevotella *Percent contribution of differences seen in PERMANOVA related to smoking.
8 Table S6. PICRUSt Analysis (see.xlsx file)
9 Table S7. ANOVA* of Richness and Diversity Data Comparing False Vocal Fold Biopsies and Vocal Fold Lesions. ANOVA CHAO SHANNON INVSIMPSON TYPE FVF**-ALL LESIONS 1.29E E ANOVA chao shannon invsimpson TYPEALL 2.20E E TUKEYHSD FVF VS CYST NODULE VS CYST POLYP VS CYST REINKE VS CYST NODULE VS FVF POLYP VS FVF 1.66E E REINKE VS FVF 1.56E E POLYP VS NODULE REINKE VS NODULE REINKE VS POLYP *TukeyHSD for multiple comparisons **FVF=False Vocal Fold
10 Table S8. PERMANOVA to Compare False Vocal Fold Biopsies and Vocal Fold Lesions Type: FVF* vs lesion BRAY-CURTIS JACCARD TYPE FVF-ALL LESIONS 1.00E E-04 RANDOM TypeAll: FVF vs Reinke vs cyst vs polyp vs nodule BRAY-CURTIS JACCARD TYPE FVF-LESION TYPES 1.00E E-04 RANDOM Pairwise PERMANOVA between all groups, Bonferroni correction for multiple comparisons BRAY-CURTIS JACCARD FVF VS REINKE FVF VS CYST FVF VS NODULE FVF VS POLYP REINKE VS CYST 1 1 REINKE VS NODULE 1 1 REINKE VS POLYP 1 1 CYST VS NODULE 1 1 CYST VS POLYP 1 1 NODULE VS POLYP 1 1 *FVF=False Vocal Fold
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