title: 16SrRNA Amplicon Sequencing of Human Gut Microbiome: Pulmonary Tuberculosis
design: Pipeline of experiment. After the DNA sample(s) was(were) delivered, we did a sample quality test first. Then we used this(those) qualified DNA sample(s) to construct BS library?Purified DNA sample, such as genomic DNA, Bacterial Artificial Chromosome or long-length PCR productions, is sheared into smaller fragments with a desired size by Covaris 3 S/E210 or Bioruptor firstly. Then the overhangs resulting from fragmentation are converted into blunt ends by using T4 DNA polymerase, Klenow Fragment and T4 Polynucleotide Kinase. After adding an A base to the 3' end of the blunt phosphorylated DNA fragments, adapters are ligated to the ends of the DNA fragments. The desired fragments can be purified though gel-electrophoresis, then selectively enriched and amplified by PCR. The index tag could be introduced into the adapter at the PCR stage as appropriate and we did a library quality test. At last, the qualified BS library would be used for sequencing.