Nextflex unique dual index barcodes

Before the library preparation, the DNA concentration of each sample was also assessed by Qubit Fluorometric Quantification (Thermo Fisher Scientific, San Jose, CA, United States). Ten nanograms of each Qubit quantified genomic DNA was sheared with a Covaris E220 instrument operating SonoLab v6.2.6 generating approximately 300 bp DNA fragments according to the manufacturer’s protocol. Between 10 and 100 ng of fragmented DNA was processed into Illumina compatible sequencing libraries using sparQ DNA Library Prep Kit (Quantabio, Beverly, MA, United States). Each library was barcoded with unique dual index sequences (NEXTFLEX^® Unique Dual Index Barcodes, BioO Scientific). Library size and amount were confirmed with a Bioanalyzer High Sensitivity DNA chip. Polymerase chain reaction primers and reagents included in the sparQ kit were used to perform PCR, and products were purified with AMPure XP beads. Equimolar libraries were pooled and subjected to Illumina NovaSeq 6000 sequencing at 2 × 150 bp (Illumina, San Diego, CA, United States). Shotgun whole metagenome sequencing was performed at the Genome Sciences and Bioinformatics Core at the Pennsylvania State University College of Medicine, Hershey, PA, United States. Illumina bcl2fastq (released version 2.20.0.422) was used to extract de-multiplexed sequencing reads.

Illumina libraries were prepared for each of three samples (G₀, G₈, and G₁₆ from cage 1:3_B) using the NEXTFLEX PCR‐free library preparation kit and NEXTFLEX Unique Dual Index Barcodes (BIOO Scientific) following the manufacturer’s instructions. The input amount of DNA was 500 ng. The ends of the DNA were repaired and adenylated. The reaction was cleaned using AMPure XP magnetic beads and Illumina barcoded adapters were ligated onto the blunt-end adenylated product. The adapter-ligated product was cleaned using AMPure XP beads. DNA quantity was measured by Qubit DNA HS assay and the fragment size assessed by Agilent Bioanalyzer 2100 DNA HS chip assay at the genomics facility of the University of Utah (GNomEx) where the libraries were sequenced on the Illumina NovaSeq with the SP flowcell 2 × 250 paired end.