16s rRNA Sequencing: Autistic
Illumina 16s rRNA protocol is a PCR-based protocol consisting of 2 PCR amplification steps each of them is followed by a clean-up step. The first PCR is called Amplicon PCR, during which the DNA template is amplified out of DNA sample using specific primers targeting the region of interest with overhang adapters attached. The second PCR step is called Index PCR during which dual indices and Illumina sequencing adapters are attached using the Nextera XT Index Kit (Illumina, USA).The KAPA Hotstar Ready Mix (KAPA Biosystems, Switzerland) was replaced with HotStar HiFidelity Polymerase Kit (QIAGEN, Germany) in both of the amplicon and indexing PCR steps. This modification required a change in the microbial DNA concentration to be increased from 5ng/l to 12.5 ng/l and change the initial step in the beginning of each PCR program according to the enzyme activation requirements to be 5 mins at 95.Degenerate primers were used in the amplicon PCR step to target the V3 and V4 regions of the bacterial rRNA and identify the microbial species. Degenerate primers are designed to allow the widest variety of V3 and V4 regions as follows:16S Amplicon (PCR Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC)Each PCR step is followed by a PCR clean-up using AMPure XP beads (Beckman Coulter, USA) to clean the amplified target from any unamplified fragments and unpaired primers.After the first PCR, fragment size is checked using the Agilent DNA 1000 Kit (Agilent Technologies, USA) with expected size of ~550 bp.Pooling and normalization of library is performed according to Illumina manufacturing instructions (Illumina, USA).