Metagenomic (whole genome seq):3K
1) DNA Extraction: The genomics DNA is extracted using the QIAamp DNA Minikit (QIAGEN, Germany) following themanufacturers instructions.2) Library Preparation and Next Generation Sequencing The preparation of the library was carried out utilizing the Nextera XT DNA Library preparation kit.I. Tagment Genomic DNA:o This step uses the Nextera transposome to tagment gDNA, which is a process that fragments DNAand then tags the DNA with adapter sequences in a single step.II. Amplify Library:o This step amplifies the tagmented DNA using a limited-cycle PCR program adding the Index 1 (i7),Index 2 (i5), and full adapter sequences to the tagmented DNA from the previous step. The indexprimers and Nextera PCR Master Mix are added directly to the 25 l of tagmented gDNA from theprevious step.III. Clean Up Libraries:IV. Check Libraries:o This step uses AMPure XP beads to purify the library DNA and remove short library fragments.o Run 1 l of library on an Agilent Technology 2100 Bioanalyzer using a Agilent DNA 1000 chip. Typicallibraries show a broad size distribution of ~2501000 bp. Various libraries can be sequenced withaverage fragment sizes as small as 250 bp or as large as 1500 bp.V. Normalize Libraries:o This process normalizes the quantity of each library to ensure more equal library representation in thepooled library.VI. Pooling Library:o Pooling libraries combines equal volumes of normalized libraries in a single tube. After pooling, diluteand heat-denature the library pool before loading libraries for the sequencing run according toIllumina manufacturing instructions.