SRX23213770 Sample1-S1 SRP483655 bp0 16S Metagenomic Sequencing Library Preparation 16S metagenomic libraries were prepared as per the Illumina 16S Metagenomic Sequencing Library Preparation protocol using Illumina primers that target the 16S V3-V4 region; 16S amplicon PCR forward primer 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG3and 16S amplicon PCR reverse primer 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3. Briefly, each 25l reaction consisted of 12.5l of 2x KAPA HiFi HotStart ReadyMix, 5l the forward primer (M), 5l of the reverse primer (M), 2.5l of DNA template (5ng/l). Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 25 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. Each experiment included sterile water as a negative control and a positive control. The amplicons were cleaned with 1.25X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol. The 16S amplicons were then indexed with unique barcode indexes (IDT for Illumina UD Indexes). Briefly, 50l reaction of indexing PCR consisted of 25l of 2x KAPA HiFi HotStart ReadyMix, 10l of the IDT for Illumina UD Index, 10l of the nuclease free water and 5l of amplicon DNA frome the first PCR. Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 8 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. The amplicons were then cleaned with 0.8X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol and eluted in 25l of nuclease free water.Library Quality control, normalization and poolingThe library was quantified using the Qubit 4.0 fluorometer (Life Technologies, Carlsbad, CA, USA), following the manufacturers protocol. Library qualification and fragment size distribution analysis was done using a Agilent fragment analyzer (Agilent Technologies, Inc. Santa Clara, CA, USA). The average fragment size of the library was expected to be 630bps.Library normalization was done by diluting each library to 4nM concentration with nuclease free water taking into consideration the average library size and concentration parameters. 5l of each normalized libraries was pipetted and then pooled into one tube SequencingThe pooled library was subsequently sequenced on a MiSeq platform (Illumina) following the manufacturers protocol. Briefly, 5ul of the 4nM pooled library was denatured and diluted to 20pM. The denatured library was spiked with sequencing control PhiX and then loaded into a MiSeq 2x300cycles V3 reagent cartridge (Illumina). The cartridge was then loaded onto the MiSeq for sequencing. The sequencing process took about 56 hours to completion and the MiSeq generated raw reads data inform of .fastq files which were used in the subsequent bioinformatics analysis. SRS20137298 RTE1 Sample1-S1 AMPLICON METAGENOMIC other Illumina MiSeq SRX23213771 Sample2-S2 SRP483655 bp0 16S Metagenomic Sequencing Library Preparation 16S metagenomic libraries were prepared as per the Illumina 16S Metagenomic Sequencing Library Preparation protocol using Illumina primers that target the 16S V3-V4 region; 16S amplicon PCR forward primer 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG3and 16S amplicon PCR reverse primer 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3. Briefly, each 25l reaction consisted of 12.5l of 2x KAPA HiFi HotStart ReadyMix, 5l the forward primer (M), 5l of the reverse primer (M), 2.5l of DNA template (5ng/l). Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 25 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. Each experiment included sterile water as a negative control and a positive control. The amplicons were cleaned with 1.25X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol. The 16S amplicons were then indexed with unique barcode indexes (IDT for Illumina UD Indexes). Briefly, 50l reaction of indexing PCR consisted of 25l of 2x KAPA HiFi HotStart ReadyMix, 10l of the IDT for Illumina UD Index, 10l of the nuclease free water and 5l of amplicon DNA frome the first PCR. Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 8 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. The amplicons were then cleaned with 0.8X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol and eluted in 25l of nuclease free water.Library Quality control, normalization and poolingThe library was quantified using the Qubit 4.0 fluorometer (Life Technologies, Carlsbad, CA, USA), following the manufacturers protocol. Library qualification and fragment size distribution analysis was done using a Agilent fragment analyzer (Agilent Technologies, Inc. Santa Clara, CA, USA). The average fragment size of the library was expected to be 630bps.Library normalization was done by diluting each library to 4nM concentration with nuclease free water taking into consideration the average library size and concentration parameters. 5l of each normalized libraries was pipetted and then pooled into one tube SequencingThe pooled library was subsequently sequenced on a MiSeq platform (Illumina) following the manufacturers protocol. Briefly, 5ul of the 4nM pooled library was denatured and diluted to 20pM. The denatured library was spiked with sequencing control PhiX and then loaded into a MiSeq 2x300cycles V3 reagent cartridge (Illumina). The cartridge was then loaded onto the MiSeq for sequencing. The sequencing process took about 56 hours to completion and the MiSeq generated raw reads data inform of .fastq files which were used in the subsequent bioinformatics analysis. SRS20137300 RTE2 Sample2-S2 AMPLICON METAGENOMIC other Illumina MiSeq SRX23213772 Sample3-S3 SRP483655 bp0 16S Metagenomic Sequencing Library Preparation 16S metagenomic libraries were prepared as per the Illumina 16S Metagenomic Sequencing Library Preparation protocol using Illumina primers that target the 16S V3-V4 region; 16S amplicon PCR forward primer 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG3and 16S amplicon PCR reverse primer 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3. Briefly, each 25l reaction consisted of 12.5l of 2x KAPA HiFi HotStart ReadyMix, 5l the forward primer (M), 5l of the reverse primer (M), 2.5l of DNA template (5ng/l). Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 25 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. Each experiment included sterile water as a negative control and a positive control. The amplicons were cleaned with 1.25X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol. The 16S amplicons were then indexed with unique barcode indexes (IDT for Illumina UD Indexes). Briefly, 50l reaction of indexing PCR consisted of 25l of 2x KAPA HiFi HotStart ReadyMix, 10l of the IDT for Illumina UD Index, 10l of the nuclease free water and 5l of amplicon DNA frome the first PCR. Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 8 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. The amplicons were then cleaned with 0.8X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol and eluted in 25l of nuclease free water.Library Quality control, normalization and poolingThe library was quantified using the Qubit 4.0 fluorometer (Life Technologies, Carlsbad, CA, USA), following the manufacturers protocol. Library qualification and fragment size distribution analysis was done using a Agilent fragment analyzer (Agilent Technologies, Inc. Santa Clara, CA, USA). The average fragment size of the library was expected to be 630bps.Library normalization was done by diluting each library to 4nM concentration with nuclease free water taking into consideration the average library size and concentration parameters. 5l of each normalized libraries was pipetted and then pooled into one tube SequencingThe pooled library was subsequently sequenced on a MiSeq platform (Illumina) following the manufacturers protocol. Briefly, 5ul of the 4nM pooled library was denatured and diluted to 20pM. The denatured library was spiked with sequencing control PhiX and then loaded into a MiSeq 2x300cycles V3 reagent cartridge (Illumina). The cartridge was then loaded onto the MiSeq for sequencing. The sequencing process took about 56 hours to completion and the MiSeq generated raw reads data inform of .fastq files which were used in the subsequent bioinformatics analysis. SRS20137299 RTE3 Sample3-S3 AMPLICON METAGENOMIC other Illumina MiSeq SRX23213773 Sample4-S4 SRP483655 bp0 16S Metagenomic Sequencing Library Preparation 16S metagenomic libraries were prepared as per the Illumina 16S Metagenomic Sequencing Library Preparation protocol using Illumina primers that target the 16S V3-V4 region; 16S amplicon PCR forward primer 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG3and 16S amplicon PCR reverse primer 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3. Briefly, each 25l reaction consisted of 12.5l of 2x KAPA HiFi HotStart ReadyMix, 5l the forward primer (M), 5l of the reverse primer (M), 2.5l of DNA template (5ng/l). Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 25 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. Each experiment included sterile water as a negative control and a positive control. The amplicons were cleaned with 1.25X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol. The 16S amplicons were then indexed with unique barcode indexes (IDT for Illumina UD Indexes). Briefly, 50l reaction of indexing PCR consisted of 25l of 2x KAPA HiFi HotStart ReadyMix, 10l of the IDT for Illumina UD Index, 10l of the nuclease free water and 5l of amplicon DNA frome the first PCR. Thermal cycling was carried out on a SimpliAmp Thermocycler (Applied Biosystems) under the following conditions: Enzyme activation at at 95 C for 3 minutes, then 8 cycles of denaturation at 95 C for 30 seconds and annealing at 55 C for 30 seconds, extension at 72C for 30 seconds, and a final extension at 72 C for 5 minutes. The amplicons were then cleaned with 0.8X AMPure XP magnetic beads (Beckman Coulter Life sciences) as per the manufacturers protocol and eluted in 25l of nuclease free water.Library Quality control, normalization and poolingThe library was quantified using the Qubit 4.0 fluorometer (Life Technologies, Carlsbad, CA, USA), following the manufacturers protocol. Library qualification and fragment size distribution analysis was done using a Agilent fragment analyzer (Agilent Technologies, Inc. Santa Clara, CA, USA). The average fragment size of the library was expected to be 630bps.Library normalization was done by diluting each library to 4nM concentration with nuclease free water taking into consideration the average library size and concentration parameters. 5l of each normalized libraries was pipetted and then pooled into one tube SequencingThe pooled library was subsequently sequenced on a MiSeq platform (Illumina) following the manufacturers protocol. Briefly, 5ul of the 4nM pooled library was denatured and diluted to 20pM. The denatured library was spiked with sequencing control PhiX and then loaded into a MiSeq 2x300cycles V3 reagent cartridge (Illumina). The cartridge was then loaded onto the MiSeq for sequencing. The sequencing process took about 56 hours to completion and the MiSeq generated raw reads data inform of .fastq files which were used in the subsequent bioinformatics analysis. SRS20137302 RTE4 Sample4-S4 AMPLICON METAGENOMIC other Illumina MiSeq