title: metagenomic shotgun sequencing of gorilla 4: prepared for culture
design: Metagenomic shotgun sequencing on prepared microbiota samples was performed at the University of Minnesota Genomics Center (UMGC). DNA samples were quantified using a fluorimetric PicoGreen assay. For a sample to pass QC, it needs to quantify greater than 0.2 ng/ul. If the samples pass QC they enter the TruSeq NexteraXT DNA library preparation queue. gDNA samples were converted to Illumina sequencing libraries using Illuminas NexteraXT DNA Sample Preparation Kit (Cat. # FC-130-1005). 1 ng of gDNA is simultaneously fragmented and tagged with a unique adapter sequence. This tagmentation step is mediated by a transposase. The tagmented DNA is simultaneously indexed and amplified 12 PCR cycles. Final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen). Truseq libraries were hybridized to a NextSeq (either Single Read or Paired End). Clustering occurs on-board where the bound library molecules are clonally amplified and sequenced using Illuminas SBS chemistry. NextSeq uses 2-color chemistry to image the clusters. Upon completion of read 1, a 7 base paired index read is performed in the case of single indexed libraries. If dual indexing was used during library preparation, 2 separate 8 or 10 base pair index reads are performed. Finally, clustered library fragments were re-synthesized in the reverse direction thus producing the template for paired end read 2. Base call (.bcl) files for each cycle of sequencing are generated by Illumina Real Time Analysis (RTA) software. The base call files are demultiplexed and then converted to index specific fastq files using the MiSeq Reporter software on-instrument.