oral metagenome in patients with oral candidiasis
Sequencing libraries were prepared according to Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 regions. Input gDNA (5 ng) was PCR-amplified using a 5x reaction buffer, 1 mM dNTP mix, 500 nM each of universal F/R primers, and Herculase II fusion DNA polymerase (Agilent Technologies). The 1st PCR conditions were: 3 min at 95C, 25 cycles of 30 sec at 95C, 30 sec at 55C, 30 sec at 72C, and a final extension of 5 min at 72C. The primers used were:- Forward: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG- Reverse: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCCThe 1st PCR product was purified with AMPure beads (Agencourt Bioscience). The purified product (10 L) was then PCR-amplified for final library construction using NexteraXT Indexed Primer with the same conditions as the 1st PCR, except for 10 cycles. The final PCR product was purified with AMPure beads, quantified using qPCR (KAPA Library Quantification kits), and qualified using TapeStation D1000 ScreenTape (Agilent Technologies).
SUB14659374
P09-posttreat-paird