2.3. Microbial Diversity Analysis

Bacterial community analysis of mixed silage was carried out at Lc-bio Technologies Co., Ltd. (Hangzhou, China) by high-throughput sequencing. DNA was extracted from the samples using an MN NucleoSpin 96 Soi DNA kit (Gene Company Limited, Beijing, China) in accordance with the kit’s instructions. The DNA obtained from each sample was subjected to two-step PCR amplification to construct a small-fragment sequencing library. For the first amplification step, the 16S rRNA gene of V3-V4 was amplified by extracting DNA as a template (primers: 314F, 5’-CCTACGGGNGGCWGCAG-3’, 805R, 5’- GACTACHVGGGTATCTAATCC-3’). PCR was carried out using a Veriti 96-well PCR instrument (9902, ABI) with a 10 µL system as follows: 50 ng of genomic DNA, 0.3 µL of Vn F (10 µmol/L), 0.3 µL of Vn R (10 µmol/L), 5 µL of KOD FX Neo Buffer, 2 µL of dNTP (2 mmol/L), 0.2 µL of KOD FX Neo, and 2.2 µL of ddH₂O. The PCR conditions were as follows: 98 °C for 30 s, 98 °C for 10 s, 54 °C for 30 s, 72 °C for 45 s, and 72 °C for 10 min 35 cycles. The Solexa PCR products were recycled from a 2.0% agarose gel and purified using an OMEGA DNA purification column. The purified products were quantified using a Quant-iT PicoGreen dsDNA assay kit in accordance with the kit’s instructions. Thereafter, the amplicons were sequenced on an Illumina HiSeq 2500 sequencing platform using the paired-end sequencing method. The original sequences obtained were spliced using FLASH software (version 1.2.11) to obtain the original tag data. The raw tags obtained were filtered using Trimmomatic software (version 0.33) to obtain high-quality clean tag data. UCHIME software (version 8.1) was then used to identify and remove chimeric sequences to obtain effective tags. The tags were binned into operational taxonomic units (OTUs) using the clustering program USEARCH (version 10.0) based on a 97% sequence similarity level [20]. The OTUs obtained were eventually used for taxonomic assignment. The representative sequences for each OTU were compared with the Silva (Release128, http://www.arb-silva.de) database to obtain taxonomic classification at the phylum, class, order, family, and genus levels. The relative abundances of taxa in the mixed silage were determined using QIIME software to compare the bacterial community composition in the mixed silage with different additives [21]. QIIME can also be used to calculate the diversity of Beta [22]. Richness and diversity indices were determined using MOTHUR software (version 1.30) to compare the bacterial diversity among different additives.