16s rRNA sequencing (V1-V3, V3-V5) of human gut microbiome of amoebiasis cases
Patient stool sample were collected from Safdarjung hospital in New Delhi after their informed consent. The stool samples were transported in ice and stored at -80C till analysis. DNA was isolated from stool sample using Qiagen mini Stool DNA isolation kit following manufacturers protocol. A bead beating step was added to the manufacturers protocol in order to ensure better DNA isolation. The isolated DNA was then stored at -20C. PCR amplification of the isolated stool DNA was performed to identification of E.histolytica infection using primers specific for E.histolytica in Srivastava et al. (F: 5GTCAGAACCACATGAAAC 3, R: 5 CGAGACCCCTTAAAGAAACCC 3; temperature: 55C). The PCR selected those patient stool DNAs which amplified the targeted region in E.histolytica thus testing positive for E.histolytica infection. METAGENOMIC ANALYSIS: Amplification and sequencing of the 16S rDNA V1-V3 & V3-V5 regions from the stool DNA of controls and patient samples positive for E.histolytica infections  were done using Illumina HiSeq 2500 Rapid/MiSeq sequencing platforms. The Universal bacterial primers were used for PCR amplification. PCR reaction volume of 25 l was set up using NEB Taq polymerase. PCR conditions were as follows: initial denaturation at 95C for 30s, followed by 30 cycles of 95C for 30s;  annealing at specific temperatures for 45s; and 68C for 30s. The final extension temperature was 72C for 5 min. The PCR products were purified using Pure link kit and were used to  build a library for sequencing on Illumina platforms. Libraries from each of the sample were tracked using a sequence barcode. Library quantification was done on Qubit 3.0.