Illumina MiSeq DNA Input 50 ng 125 ng Amplicon Generation 1 Round Fusion PCR 2 PCR rxns sample 2 Round Tailed PCRs 1 PCR rxn sample Qiagen Hot Star HiFidelity Polymerase 1U ID: 1043069
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1. 454 GS FLXIon Torrent PGMIllumina MiSeqDNA Input50 ng12.5 ngAmplicon Generation 1 Round Fusion PCR(2 PCR rxns/sample)2 Round Tailed PCRs(1 PCR rxn/sample)Qiagen Hot Star HiFidelity Polymerase (1U)Kapa HiFidelity Hot StartPolymerase (0.5 U)Primer F: Adapter A + Barcode + 8FPrimer R: 338R + Barcode + Adapter B Primer F: Adapter A + Barcode + 8FPrimer R: 338R + Adapter trP11 Round PCR Primer F: Overhang + 8FPrimer R: 338R + Overhang2 Round PCR Primer F: Nextera Index 1 Primer R: Nextera Index 2 Thermocycler Conditions35 Cycles, Annealing T°C: 50°C25 Cycles, Annealing T°C: 55°CAmplicon Purification 2% Agarose E-GelMagnetic BeadsMiSeq1 = MiSeq235 Cycles, Annealing T°C: 50°C2% Agarose E-Gel25 Cycles, Annealing T°C: 50°C454 GS FLX35 Cycles, Annealing T°C: 55°CPGM1PGM2PGM3
2. GS FLXPGMMiSeqRaw sequencesQuality controlTrimmed and filtered readsCombined fna filesInitial ProcessingUPARSEQIIMEQIIME1QIIME2QIIME3QIIME4UPARSE1UPARSE2De novo OTU pickingOpen referenceOTU pickingChimera detectionTaxonomy assignment Alpha and beta diversity analysesOTU pickingChimera detectionTaxonomy assignment Alpha and beta diversity analysesPerformed for each platform separatelyChimera detectionError Suppression/ DereplicationTaxonomy assignment Alpha and beta diversity analysesDADA2 (Illumina only)
3. Significantly different (p<0.05)Not significantly different (p>0.05)GSFLEXMiSeq10.0150.060.180.0150.0150.015GSFLEXMiSeq20.030.0750.0750.030.0150.03MiSeq1MiSeq2111111PGM1MiSeq20.1350.060.6910.5551PGM1MiSeq10.060.1210.6450.0450.105PGM1GSFLEX0.0150.01510.2250.1950.015PGM2PGM30.0150.030.0150.0150.015PGM2MiSeq2111111PGM2MiSeq1111110.15PGM2GSFLEX0.0150.0150.330.030.0150.015PGM2PGM10.360.4351111PGM3PGM10.0150.0150.0750.0150.1050.015PGM3GSFLEX111111PGM3MiSeq10.0150.0150.0150.0150.0150.015PGM3MiSeq20.0450.030.0150.0150.0150.015QIIME1QIIME2QIIME3QIIME4UPARSE1UPARSE2PLATFORMGSFLEXMiSeq10.0750.1950.090.1050.030.03GSFLEXMiSeq211110.360.93MiSeq1MiSeq20.0150.0150.1050.2250.0750.015PGM1MiSeq2111111PGM1MiSeq10.0150.015110.7050.195PGM1GSFLEX111111PGM2PGM30.180.1650.030.030.1050.06PGM2MiSeq2111111PGM2MiSeq10.2550.2550.0150.0150.0150.015PGM2GSFLEX110.090.28511PGM2PGM10.0150.031111PGM3PGM10.0150.031111PGM3GSFLEX0.30.2250.0150.0150.0150.015PGM3MiSeq1110.060.030.0750.09PGM3MiSeq20.0150.0150.0150.030.0150.015Figure 3(A)(B)ANALYSIS PIPELINEPLATFORMPhylogenetic DiversitySpecies Richness
4. QIIME1ANOSIM R=0.4946, P=0.001, PERMANOVA F=7.2165, P=0.001, QIIME2ANOSIM p=0.001, R=0.4998PERMANOVA p=0.001, F=7.3045 QIIME3ANOSIM p=0.001, R=0.5015 PERMANOVA p=0.001, F=8.4217 QIIME4ANOSIM p=0.001, R=0.5052PERMANOVA p=0.001, F=8.6540UPARSE1ANOSIM p=0.001, R=0.5768PERMANOVA p=0.001, F=10.1628 UPARSE2ANOSIM p=0.001, R=0.5554 PERMANOVA p=0.001, F=9.4836 Control-ChallengedVaccinatedVaccinated - ChallengedPrebioticsPrebiotics-ChallengedMonte Carlo p < 0.01M2= 0.387 Monte Carlo p < 0.01M2= 0.482Monte Carlo p < 0.01M2= 0.345GS FLEX – MiSeq1 GS FLEX – PGM1MiSeq– PGM1(A)(B)
5. QIIME1QIIME2QIIME3QIIME4UPARSE1UPARSE2GS FLEXIllumina1Illumina2PGM1PGM2PGM3
6. ActinobacteriaBacteroidetesFirmicutesFusobacteriaOtherCyanobacteriaChlorobiChloroflexiProteobacteriaVerrucomicrobiaTenericutesThermiTM7De novoOpen refUPARSE***QIIMEBifidobacterium adolescentisBifidobacterium spp.Lactobacillus Lactobacillus_agilis Lactobacillus_iners Lactobacillus_reuteri Lactobacillus_spp. Lactobacillus_zeae Tepidimicrobium_spp. Thermacetogenium_spp. Tindallia_Anoxynatronum_spp. Tissierella_Soehngenia_spp.
7. DADA2.DADA2/DADA2.QIIMEDADA2.DADA2/QIIME.QIIMEDADA2.QIIME/ QIIME.DADA2QIIME.QIIME/ QIIME.DADA2(A)(B)(D)ControlPrebioticsVaccinated(C)Control_run1Control_run2Vaccinated_run1Vaccinated_run2Prebiotics_run1Prebiotics_run2Vaccinated