Alteration of the fecal microbiome in patients with gallstones: potential relationship with post-cholecystectomy diarrhea

16S ribosomal RNA amplification and sequencing data from fecal samples

1) Sample Preparation
For library construction, DNA/RNA is extracted from a sample. After performing quality control (QC), qualified samples proceed to library construction.
2) Library Construction
The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process.
Adapter-ligated fragments are then PCR amplified and gel purified.
3) Sequencing
For cluster generation, the library is loaded into a flow cell where fragments are captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment is then amplified into distinct, clonal clusters through bridge amplification. When cluster generation is complete, the templates are ready for sequencing. Illumina SBS technology utilizes a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all 4 reversible, terminator-bound dNTPs are persent during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies. The result
is highly accurate base-by-base sequencing that virtually eliminates sequence-context-specific errors, even within repetitive sequence regions and homopolymers.
4) Raw data
Sequencing data is converted into raw data for the analysis.