gut metagenome9
Sequencing libraries were prepared using Illuminas TruSeq Nano DNA LT Library Prep Kit.1) End repair was performed on the amplified products. Using End Repair Mix 2 from the kit, excess bases at the 5 end of the DNA sequence were removed, and a phosphate group was added to that end. At the same time, missing bases at the 3 end were added;2) Add an A base to the 3 end of the DNA sequence. The purpose of this step is to prevent self-ligation of the DNA fragments and to ensure that the target sequence can be ligated to the sequencing adapter (the 3 end of the sequencing adapter has a protruding T base);3) Attach a sequencing adapter containing a library-specific tag (also known as an index sequence) to the 5 end of the sequence. This allows the DNA molecule to be firmly anchored to the flow cell;4) Use BECKMAN AMPure XP Beads to remove adapter-self-ligated fragments via magnetic bead purification, thereby purifying the adapter-tagged library;5) PCR amplification of the DNA fragments with attached adapters is performed to enrich the sequencing library template. Subsequently, the enriched library product is purified again using BECKMAN AMPure XP Beads;6) Final fragment selection and purification of the library are performed via 2% agarose gel electrophoresis.