Whole genome sequencing of Staphylococcus aureus: Isolates associated with mastitis
Using the standard Illumina TruSeq Nano DNA LT library preparation protocol (Illumina TruSeq DNA Sample Preparation Guide) to construct the required genomic sequencing library. The main experimental process is as follows (Kit: TruSeqTM DNA Sample Prep Kit):(1) DNA Fragmentation: DNA is fragmented using Covaris to generate smaller pieces, which are then purified with magnetic beads to select the target fragments;(2) DNA End Repair: DNA fragments with overhanging ends are repaired through the action of the 3'-5' exonuclease and polymerase in the End Repair Mix;(3) Adding an 'A' base at the 3' end: A single 'A' base is added to the 3' end of the repaired DNA fragments, while the 3' end of the adapters contains a single 'T', ensuring that the DNA fragments and adapters can connect through complementary pairing of 'A' and 'T', preventing the DNA insert fragments from connecting to each other during the adapter ligation process;(4) Adapter Ligation: Under the action of ligase, the labeled adapters are incubated with the DNA fragments to link them;(5) Purifying the ligation products: Magnetic beads are used to purify the ligation products, removing free and self-ligated adapter sequences;(6) Enrichment of DNA fragments: PCR is utilized to selectively enrich the DNA fragments that are ligated at both ends to adapters while amplifying the DNA library;(7) Library Verification: The library is quantified using Pico Green; the quality of the PCR-enriched fragments is assessed using the Agilent Bioanalyzer 2100 to verify the fragment size and distribution of the DNA library;(8) Normalizing and pooling the library: After normalizing multiplexed DNA libraries to 10 nM, they are mixed in equal volumes;(9) Sequencing: The mixed library (10 nM) is gradually diluted and quantified to 4-5 pM before sequencing.