Vahts universal dna library prep kit

Bacterial genomic DNA of A. baumannii strains were extracted using a HiPure Bacterial DNA Kit (MAGEN). The libraries were created using the VAHTS™ Universal DNA Library Prep kit for Illumina. Whole genome sequencing was carried out through an Illumina Hiseq 2500 system to obtain 2 × 150 bp reads. The sequence quality of the reads was evaluated using FastQC. Processed reads were de novo assembled into contigs with CLC Genomics Workbench 10.1 (CLC Bio, Aarhus, Denmark), and genomes were annotated by NCBI Prokaryotic Annotation Pipeline (PGAP).

Next generation sequencing library preparations were constructed following the manufacturer’s protocol (VAHTS Universal DNA Library Prep Kit for Illumina). The fecal samples from Cubs, Young, Adults, and Old pandas (three samples per group) were analyzed where 200 ng genomic DNA was randomly fragmented to <500 bp by sonication (Covaris S220) with the setting parameters of peak power: 175 w, duty factor: 10%, Cycles/Burst: 200, and times: 50 s. The fragments were treated with End Prep Enzyme Mix for end repairing, 5’ Phosphorylation and dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using VAHTSTM DNA Clean Beads, and fragments of ~470 bp were recovered. Each sample was then amplified by PCR for eight cycles using P5 and P7 primers, with both primers carrying sequences that can anneal with flowcell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) quantified by Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), and sequenced using an Illumina HiSeq/Novaseq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA).

Two purified sponge cell fractions I and II were pooled for metagenomic sequencing. Library preparations were constructed following the manufacturer’s protocol (VAHTS Universal DNA Library Prep Kit for Illumina). Replicates were applied. The PCR products were cleaned up using VAHTSTM DNA Clean Beads, validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2 × 150 paired-end configuration on Illumina HiSeq instrument.