CR_2018_metagenome
Library preparation was performed using the NEXTflexTM DNA Sequencing kit for Ion Platforms (PerkinElmer, Waltham, MA, USA) according to the manufacturers protocol, BI00 Scientific V15.12, December 2015. Briefly, DNA was fragmented using the Covaris S2 focused ultrasonicator (Covaris, Inc.; Woburn, MA, USA) using a 10% duty cycle, with 5% intensity, 200 cycles/ burst in two cycles of 60sec treatment time. Sheared DNA was purified using 1.8x volume Agencourt AMPure XP reagent (Beckman Coulter, Brea, CA, USA). The fragmented DNA was end-repaired at 22C for 30min and purified using 1.8x volume Agencourt AMPure XP reagent (Beckman Coulter). End-repaired DNA was blunt-end ligated to IonCode Barcode Adapters at 22C for 15min and purified, using 1.8x volume Agencourt AMPure XP reagent (Beckman Coulter). The adapter-ligated library was size selected on the Pippen Prep (Sage Science, Beverly, MA, USA) using 2% dye-free Gel cassettes with marker L, to retain DNA fragments of ~275bp. The adapter-ligated, size-selected library was amplified across seven polymerase chain reaction (PCR) cycles. The amplified libraries were purified using 1.8x volume Agencourt AMPure XP reagent (Beckman Coulter) and assessed for fragment size distribution on the LabChip GXII Touch (PerkinElmer, Waltham, MA, USA), using the X-mark chip and HT DNA NGS 3K reagent kit according to the manufacturers protocol; CLS145098 Rev. E. Library fragments were quantified using the Library TaqMan Quantitation Kit according to the manufacturers protocol, MAN0015802, Rev. B.0. This quantitative PCR amplification was performed on the StepOnePlus Real-time PCR system (ThermoFisher Scientific). The molarity of libraries and the respective barcodes are noted relative to sample identifiers