16S rRNA- Seq of Gut Microbiota: Parkinson's Disease
Sequencing libraries were constructed by a two-step PCR. Round 1 PCR -The 16S rRNA gene V3-V4 regions were first amplified using region-specific primers, genomic DNA was amplified for 26 cycles. Round 2 PCR for indexing- round 1 PCR amplicons were amplified for 10 cycles to add Illumina sequencing barcoded adaptors. The libraries were normalized and pooled for multiplex sequencing. Then diluted to 2 nM final concentration using Resuspension Buffer.  The normalized sample was denatured for 5 minutes using 0.2 N NaOH and neutralized by HT1 Buffer. Denatured libraries were further diluted 13 pM concentration for loading.