title: 16s RNA amplicon V1_V3 and V3_V4 June rep A
design: PROTOCOL 16S Metagenomic Sequencing Library Preparation - Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System Part # 15044223 Rev. B METHOD Firstly, two primer pairs were designed and ordered according to customer's wishes. After receiving the DNA samples the concentrations were measured using Nanodrop and Qubit. Samples which did not yield at all or yielded under 3,125 ng/ul were excluded from the PCR step. Due to the scarce amount of sample material it was not possible to check the quality of all the samples on a gel. For each sample two PCR's were performed to amplify the two previously chosen 16S rRNA regions V1-V3 and V3-V4. After the PCR each amplified sample was purified using Ampure XP beads and run with Bioanalyzer HS DNA chip to check the PCR amplification. In case of no amplification, the PCR reaction was performed anew. All samples which did successfully yield a band for the PCR product were taken into the indexing step. The PCR products of the two different regions, V1-V3 and V3-V4, were pooled per sample prior to indexing in a way that the concentrations of both products stayed even. After the indexing the prepared libraries were purified again using Ampure XP beads.  Lastly, before pooling and sequencing, the libraries were quantified using Qubit (dsDNA HS) and the size of each library was estimated using Bioanalyzer (DNA HS)