TITLE: U Michigan - DNA Extraction for Illumina 16S rRNA Extraction
AUTHORS: Vincent Young

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">This protocol is for the submission of tissue/fecal samples for DNA extraction and subsequent processing to generate libraries for 16S rRNA sequencing, which can be used for bacterial community analysis and detect variations in the microbiota under differing conditions.   </div><div class = "text-block">Up to 250 uL or 0.25 g of sample is added to each well of the Bead plate provided by the MMPC.  Plates are properly packaged and shipped to the MMPC and processed using the Qiagen MagAttract PowerMicrobiome kit DNA/RNA kit (Qiagen, catalog no. 27500-4-EP) on the EpMotion 5075 (Eppendorf) liquid handler.  DNA is lysed using mechanical bead beating and extracted using magnetic bead technology.  Extracted DNA is then used to generate 16S rRNA libraries for community analysis.  The process used for library generation has been previously described by Seekatz et al. (1).   Briefly, barcoded dual-index primers specific to the V4 region of the 16S rRNA gene amplify the DNA (2).  PCR reactions are composed of 5 µL of 4 µM equimolar primer set, 0.15 µL of AccuPrime Taq DNA High Fidelity Polymerase, 2 µL of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 µL of PCR-grade water, and 1 µL of DNA template.  The PCR conditions used consisted of 2 min at 95°C, followed by 30 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 5 min, followed by 72°C for 10 min.  Each PCR reaction is normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001).  The normalized reactions are pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873).  The Agilent Bioanalyzer is used to confirm the size of the amplicon library (~399 bp) using a high-sensitive DNA analysis kit (catalog no. 5067-4626).  Pooled amplicon library is then sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1/read 2 and index primers added to the reagent cartridge.  The “Preparing Libraries for Sequencing on the MiSeq” (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 pM, spiked with 15% PhiX to create diversity within the run.  FASTQ files are distributed to the client when the 2 x 250 bp sequencing completes.</div><div class = "text-block"><span style = "font-weight:bold;">References:</span></div><div class = "text-block"><span>1. </span><span style = "font-weight:bold;">Seekatz AM, Theriot CM, Molloy CT, Wozniak KL, Bergin IL, Young VB.</span><span> 2015. Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease. Infect Immun </span><span style = "font-weight:bold;">83</span><span>:3838-3846. 10.1128/IAI.00459-15.</span></div><div class = "text-block"><span>2. </span><span style = "font-weight:bold;">Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD.</span><span> 2013. Development of a dual- index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol </span><span style = "font-weight:bold;">79</span><span>:5112–5120. 10.1128/AEM.01043-13.</span></div></div>

[STEPS]
?. Briefly centrifuge the PowerMag Glass Bead plate to collect beads at the bottom of the well.  In a clean hood, add a maximum of 0.25g or 250 uL of sample to the PowerMag Glass Bead plate and seal with mat.  Reserve at least three wells in the bead plate for controls.  Maximum number of samples per plate is 93.  Clearly label plates with PI, Reference ID, and date.
?. Use these shipping directions to prepare the Bead plates for shipping (use the same instructions as PCR plates).
?. Fill out submission form.
?. Send electronic plate map and shipment tracking information to msmblcore@umich.edu.
?. Ship on dry ice to: Attention: April Cockburn Mouse Metabolic Phenotyping Centers University of Michigan Medical School Internal Medicine/Infectious Diseases 1500 MSRB1 1150 W. Medical Center Drive Ann Arbor, MI 48109-5666 Please include reference ID on package documentation.