SRX373516 UP_b1_454_DNA SRP032651 DNA libraries for pyrosequencing were prepared from extracted DNA from the October and February samples, which were nebulized to a size range of 500-1000bp. Each library was prepared from 500 ng of DNA. Library preparation was performed using the GS FLX Titanium Rapid Library Preparation Kit (Roche Applied Science), following the manufacturer’s protocol. SRS498874 Sludge_b1_October UP_b1_454_DNA WGS METAGENOMIC RANDOM 0 0 Technical Read Adapter 1 1 Application Read Forward 5 454 GS FLX SRX373517 UP_b1_GAIIx_DNA SRP032651 Extracted DNA from the October samples was sheared on a Covaris S2 instrument to 300bp following the manufacturers recommendation. For each sample, 2.5 μg of DNA was used in a total volume of 120 μL of TE buffer. The sheared DNA samples were subjected to Illumina paired-end library preparation using a Beckman SPRIworks Fragment Library System and Beckman Fragment Library Kit I reagents. Ten microliters of PE Adapter Oligo Mix (Illumina) were loaded onto the instrument for each sample. Library preparation was performed according to the pre-programmed Illumina library preparation method. Size-selection on the SPRIworks was skipped and performed separately after PCR amplification. After library preparation, samples were removed from the SPRIworks and quantitated using Picogreen fluorescent dye (Invitrogen). The concentration of each library was normalized to 10 μg/μL. For each library, 4 independent PCR reactions were set-up consisting of the following components: 1 μL of normalized library, 0.5 μL of PCR PE Primer 1.0 (Illumina), 0.5 uL of PCR PE Primer 2.0 (Illumina), 23 μL of water, and 25 μL of 2x Phusion PCR Master Mix (Finnzymes). Libraries were amplified for 7 cycles using the following cycling parameters: 1= 98°C 0:30 2= 98°C 0:10 3= 65°C 0:30 4= 72°C 0:30 5= Goto 2 repeat 7x 6= 72°C 3:00 7= 10°C Hold After amplification, the 4 PCR reactions for each library were pooled, purified on a PCR Purification column (Qiagen), and eluted in 30 μL of buffer EB (Qiagen). Amplified libraries were size-selected on a Pippin Prep instrument (Sage Science) by using a 2% agarose cassette with ethidium bromide. Sizing was performed by selecting a narrow peak at 370bp +/-10%. SRS498874 Sludge_b1_October UP_b1_GAIIx_DNA WGS METAGENOMIC RANDOM 151 0 Application Read Forward 1 Illumina Genome Analyzer IIx SRX373519 UP_b1_H2000_DNA SRP032651 Extracted DNA from the October samples was sheared on a Covaris S2 instrument to 300bp following the manufacturers recommendation. For each sample, 2.5 μg of DNA was used in a total volume of 120 μL of TE buffer. The sheared DNA samples were subjected to Illumina paired-end library preparation using a Beckman SPRIworks Fragment Library System and Beckman Fragment Library Kit I reagents. Ten microliters of PE Adapter Oligo Mix (Illumina) were loaded onto the instrument for each sample. Library preparation was performed according to the pre-programmed Illumina library preparation method. Size-selection on the SPRIworks was skipped and performed separately after PCR amplification. After library preparation, samples were removed from the SPRIworks and quantitated using Picogreen fluorescent dye (Invitrogen). The concentration of each library was normalized to 10 μg/μL. For each library, 4 independent PCR reactions were set-up consisting of the following components: 1 μL of normalized library, 0.5 μL of PCR PE Primer 1.0 (Illumina), 0.5 uL of PCR PE Primer 2.0 (Illumina), 23 μL of water, and 25 μL of 2x Phusion PCR Master Mix (Finnzymes). Libraries were amplified for 7 cycles using the following cycling parameters: 1= 98°C 0:30 2= 98°C 0:10 3= 65°C 0:30 4= 72°C 0:30 5= Goto 2 repeat 7x 6= 72°C 3:00 7= 10°C Hold After amplification, the 4 PCR reactions for each library were pooled, purified on a PCR Purification column (Qiagen), and eluted in 30 μL of buffer EB (Qiagen). Amplified libraries were size-selected on a Pippin Prep instrument (Sage Science) by using a 2% agarose cassette with ethidium bromide. Sizing was performed by selecting a narrow peak at 370bp +/-10%. SRS498874 Sludge_b1_October UP_b1_H2000_DNA WGS METAGENOMIC RANDOM 202 0 Application Read Forward 1 1 Application Read Reverse 102 Illumina HiSeq 2000