title: 16s rRNA sequencing (V1-V3, V3-V5) of human gut microbiome of amoebiasis cases
design: 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.