trn-L P6 loop amplicon sequencing of horse feces
We PCR amplified the trnL-UAA P6 loop chloroplast gene region using g region 5-GGGCAATCCTGAGCCAA-3 and h region 5-CCATTGAGTCTCTGCACCTATC-3 primers17. Both primers consisted of a pool of 7 salt-free primers mixed in equal amounts containing between one and seven random bases (N) at the 5 end to increase diversity of amplicons and an adapter sequence to allow incorporation of barcodes in a second PCR. Prior to conducting PCR amplification on the entire set of samples, we first identified suitable PCR conditions by comparing amplification using the conditions detailed below but using either 40 ng or 80 ng of template, 25 or 30 amplification cycles, and an annealing temperature of 60C or 62C. Based on visualization on 2% agarose gels, we determined that using 40ng of template, 30 amplification cycles, and an annealing temperature of 62C performed best among those conditions. Co-loading of a 25-766bp ladder (Quick-Load Low Molecular Weight DNA Ladder, New England Biolabs) was also used to determine that most amplicons (including primer sequences) were approximately 200bp long. A set of 95 samples (which included 85 samples with paired metagenomic data) and 1 negative control (molecular grade water) were then amplified using PCR with the following reagents: 5 ?L 5X KAPA HiFi HotStart Fidelity Buffer (KAPA Biosystems, USA), 0.75 ?L g primer (10 ?M), 0.75 ?L h primer (10 ?M), 0.75 ?L dNTPs (10 mM), 0.5 ?L KAPA HiFi HotStart Polymerase (0.5 U), 9.25 ?L ddH2O, and 8 ?L template DNA (standardized to 10 ng/ul). Thermocycling conditions were 95C for 2 minutes, followed by 30 cycles of 98C for 20s, 62C for 15s, 72C for 15s, followed by a 2-minute final extension at 72C.  After confirming successful PCR amplification by visualizing 5ul of product on a 2% agarose gel, the remainder was purified using AMPure XP Magnetic Beads following manufacturer protocols with a 1.8X bead to template ratio (Beckman Coulter, Inc.). Ninety-six pairs of Unique Dual Indices (UDI, IDT for Illumina Set A) and P5/P7 sequencing tags were then added to the amplicons in a second PCR amplification. Reagents for this second PCR consisted of: 5?L 5X KAPA HiFi HotStart Fidelity Buffer, 1.25 ?L Forward Primer (10 ?M), 1.25 ?L Reverse Primer (10 ?M), 0.75 ?L dNTPs (10 mM), 0.5 ?L KAPA HiFi Polymerase (0.5 U), 10.25 ?L H2O, and 6 ?L of purified first round PCR product. Thermocycling conditions were 98C for 45s, followed by seven cycles of 98C for 20s, 63C for 20s, and 72C for two minutes. Products were again purified using AMPure XP magnetic beads (1.8X), visualized on a 2% agarose gel, and quantified using the Quant-it BR kit (2 ul of template). Samples were then pooled in equal amounts (100 ng/sample) into a single library, with the exception of the negative control for which 5.2 ul was added (which corresponded to the average volume added per sample). This library was quantified using absorbance with a Take3 Trio microplate adapter (averaged from 8 2ul replicates), diluted to ~5 ng/ul using molecular grade water, and re-quantified using a Quant-it HS kit (10 ul of template, 8 replicates). The library concentration in nM was then estimated assuming an average library size of 242bp (inferred from the dominant peak on an Agilent TapeStation D1000 ScreenTape) and diluted to 1 nM using 10 mM Tris-HCl (pH 8.5). This 1 nM library was further diluted to 50 pM using Tris-HCl and combined 20:1 with 50 pM PhiX Control v3 (Illumina, San Diego, USA). The library (20 ul input) was sequenced on a local Illumina iSeq 100 sequencer with a v1 300-cycle Reagent Kit following the iSeq 100 Sequencing Guide, and demultiplexed FASTQ files created using the Local Run Manager Generate FASTQ Analysis Module V2.0.1.