sequencing

The DNA libraries required for next-generation sequencing were prepared using the Illumina® Nextera XT DNA Library Preparation Kit (96 Samples).
As the first step of the study, the V3-V4 hypervariable region of the bacterial 16S rRNA gene coding region was amplified from the frozen DNA samples. The following primer pair was used for amplification: 16SBact-0341 Fwd (5ꞌ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and 16SBact-0785 Rev (5ꞌ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). The amplicons were purified with AMPure XP (Beckman Coulter, Inc., CA, USA) magnetic beads. The purified DNA fragments were diluted to a concentration of 0.5 ng/µl and then individually labeled with Nextera XT Index Kit v2 Set D (Illumina, USA) indexes (1-1 i5 and i7 index per sample) according to the manufacturer's instructions. The exact DNA concentration of the indexed amplicons was determined using the Qubit® dsDNA HS Assay (Thermo Fisher Scientific, USA) fluorimetric method, and the average fragment length of the samples was determined using the Agilent Bioanalyzer (Agilent, CA, USA). Then, the molar concentration of each sample was calculated using the following formula:
Molarity (nM)=(ng/ul x 10^6)/(660 g/mol x average library size (bp))
Each sample was diluted to a concentration of 4 nM, then 20-20 µl were measured into a sterile eppendorf tube to prepare the final DNA library pool. In the case of samples where the DNA concentration after purification did not reach 4 nM, the solution was added to the DNA pool in concentrated form without dilution. The pool concentration was checked using the Qubit® dsDNA HS Assay (Thermo Fisher Scientific, USA) method and diluted to 4 nM if necessary. 5 µl of the DNA pool was denatured with an equal volume of 0.2 M NaOH solution for 5 min, then the reaction was stopped with 990 µl of HT1 buffer. 5 µl of PhiX solution diluted to 4 nM (PhiX Control v3, Illumina, USA) was denatured in the same way as the DNA pool. The final library to be sequenced was finally adjusted to a concentration of 6 pM with 35% PhiX as follows: 117 µl denatured DNA pool + 63 µl denatured PhiX + 420 µl HT1 buffer. The library thus prepared was sequenced on an Illumina MiSeq (Illumina, USA) platform using the MiSeq Reagent Kit v3 (600 cycles).