Truseq nano lt library preparation kit

Genome sequencing was performed with Illumina technology (Illumina, San Diego, CA). Two hundred nanograms of genomic DNA quantified using a Qubit fluorometer (Life Technologies, Foster City, CA) were sheared to a peak size of 580 bp with a Covaris M220 Focused Ultrasonicator (Covaris, Woburn, MA). Thereafter, genomic sequencing libraries were prepared using a TruSeq Nano LT library preparation kit (Illumina) using indexed adapters. The quality and quantity of the libraries were assessed on a Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA) using a DNA 1000 chip. Eight libraries with different barcodes were pooled into one sequencing lane, aiming at fivefold genomic coverage for each sequenced accession. Sequencing was done on the Illumina HiSeq 2500 instrument using v4 sequencing chemistry and a 2 × 125 nt paired‐end sequencing recipe.

Exocellular DNA (vesicles, viruses, or free DNA) metagenomic libraries were prepared using a robotic liquid handling protocol (epMotion, Eppendorf, Hamburg, DE) with a TruSeq Nano DNA library preparation kit (catalog number 15041110; Illumina); a DNA input per sample of 2 ng μL⁻¹ sheared to an average size of 350 bp utilizing a Covaris M220 focused ultrasonicator according to the manufacturer’s recommendations, with modifications to the shear time to target 350 bp; and Microtube-50 AFA fiber tubes. (For some selected samples that had bimodal DNA size distributions, two sequencing libraries were prepared, one with DNA shearing and one without DNA shearing.) Metagenome sequencing libraries were prepared using Illumina’s TruSeq Nano LT library preparation kit and sequenced on an Illumina Nextseq500 system, using the V2 high-output 300-cycle reagent kit, with the addition of 1% of a PhiX control.