title: Illumina-Miseq of Mus musculus: feces
design: The V4 region of the 16srRNA gene was amplified from each sample using the Dual-indexing sequencing strategy.  Sequencing was done on the Illumina MiSeq platform, using a MiSeq Reagent Kit V2 500 cycles (Cat# MS-102-2003), according to the manufacturers instructions with modifications found in the Schloss SOP. Accuprime High Fidelity Taq (Life Cat # 12346094 ) was used instead of Accuprime Pfx supermix.  PCR products are visualized using an E-Gel 96 with SYBR Safe DNA Gel Stain, 2% (Life technologies cat# G7208-02).  Libraries are normalized using Life technologies SequalPrep Normalization Plate Kit (cat # A10510-01) following the manufactures protocol for sequential elution.  The concentration of the pooled samples is determined using Kapa Biosystems Library Quantification kit for Illumina platforms (KapaBiosystems KK4824).  The sizes of the amplicons in the library are determined using the Agilent Bioanalyzer High Sensitivity DNA analysis kit (cat# 5067-4626).  The final library consists of equal molar amounts from each of the plates, normalized to the pooled plate at the lowest concentration. Libraries are prepared according to Illuminas protocol for Preparing Libraries for Sequencing on the MiSeq (part# 15039740 Rev. D) for 2nM or 4nM libraries. If the library concentration is below 1nM an alternative method is used for denaturation. (2).  The final load concentration is 4pM with a 10% PhiX spike to add diversity.  Sequencing reagents are prepared according to the Schloss SOP, custom read 1, read 2 and index primers are added to the reagent cartridge.  FASTQ files are generated for paired end reads.