Total compost DNA was extracted from triplicate compost subsamples per treatment replicate. Extraction was done using the PureLink™ Microbiome DNA Purification Kit (Catalog number: A29790) as per the manufacturer’s instructions. DNA quality and concentration per sample were confirmed using NanoDrop (Maestrogen) and visually under 2% agarose gel. The extracted compost DNA was shipped under dry ice to the Molecular Research DNA Lab (www.mrdnalab.com , Shallowater, TX, USA) for downstream processing.
The 16S rRNA gene V4 variable region PCR primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (GGACTACNVGGGTWTCTAAT) were used in PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). The following conditions were used for 16S rRNA gene amplification: Initial denaturation heating at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 40 s, and extension at 72 °C for 1 min, and final elongation at 72 °C for 10 min. After amplification, PCR products were checked in 2% agarose gel to determine the amplification success and the bands’ relative intensity. Equimolar quantities of PCR amplicons obtained from 16 individual composts were multiplexed using unique indices, pooled, and sequenced using Illumina MiSeq next-generation technology at MR DNA (www.mrdnalab.com , Shallowater, TX, USA).
The 16S rRNA gene V4 variable region PCR primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (GGACTACNVGGGTWTCTAAT) were used in PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). The following conditions were used for 16S rRNA gene amplification: Initial denaturation heating at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 40 s, and extension at 72 °C for 1 min, and final elongation at 72 °C for 10 min. After amplification, PCR products were checked in 2% agarose gel to determine the amplification success and the bands’ relative intensity. Equimolar quantities of PCR amplicons obtained from 16 individual composts were multiplexed using unique indices, pooled, and sequenced using Illumina MiSeq next-generation technology at MR DNA (