title: ITS sequencing of PDAC mouse stool
design: The sequencing libraries were prepared using a two-step PCR method using the primer set ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS2 (5-GCTGCGTTCTTCATCGATGC-3). The first PCR (25-cycle) uses 25ng of DNA to amplify the target region, where the PCR primers have overhang adapter sequence necessary for the second PCR step.  After purification,  the amplicon from the first step is amplified with 8 cycles of PCR using the Nextera Index Kit (Illumina Inc.), which uses primers that target the overhang adaptor sequence added during the first round of PCR.  The second round PCR adds one of 384 different combinations of indexed tags to each sample which allows pooling of libraries and multiplex sequencing.  Prior to pooling, each individual samples amplified DNA is visualized on a Tapestation 4200 D1000 tape (Agilent Technologies) for expected amplicon size, purity and concentration. Validated libraries are pooled equal molar in a final concentration of 4nM in TrisHCI 10 mM, pH 8.5, before 2 x 300 cycle sequencing on a MiSeq (Illumina, Inc.).