Host Microbe Systems Biology Core Tupper Hall Room 3439 (530) 754-7850 Director: Satya Dandekar, Ph.D. Associate Director: Loubna Tazi, Ph.D. Matt Rolston Manager: mrrolston@ucdavis.edu http://www.ucdmc.ucdavis.edu/medmicro/hmsbcore/index.html
Why do we utilize 16S sequencing? All bacteria have a 16S gene Highly conserved because it is necessary for ribosomes to translate mRNA Because this gene has changed very little over time we can construct phylogenies to examine the evolution of bacteria and their relationships to one another This conserved nature also allows us to construct universal primers that will amplify gene sequences across many different bacterial groups Sequence information can be compared to known reference databases and identification can be accurately described down to the genus level (or species, or sub-species, depending on who you ask)
What 16S Sequencing IS and what it IS NOT IS IS NOT Tool for Generating the Final Tool for Generating New Answer Hypotheses A standalone technique that Powerful when combined with requires no other validation other techniques like qPCR and Gene Expression data A purely quantitative tool that Qualitative with some will solve all of your problems quantitative elements and wrap them up with a nice depending on experimental little bow design The Beginning The End 16S sequencing is a valuable resource IF you understand its limitations
Human Health and Disease Primate Models Development Studies Nutrition Studies Mouse Models 16S Microbial Agriculture Studies Analysis Insect Models The Built Environment Environmental Communities
UC Davis Host Microbe Systems Biology Core Typical 16S Microbial Analysis Workflow DNA Extraction Qiagen (MoBio™) Purification Kits Library Amplification of 16S region Construction Paired end barcoding Illumina MiSeq Sequencing Potential output: 10 – 15 Gb QIIME: Rob Knight, UC San Diego Data Analysis Mothur: Pat Schloss, Univ. of Michigan DADA2: BCallahan and SHolmes, Stanford 1. Sample Diversity 2. Community Structure 3. Conditional Modulations
Why the MiSeq for 16S?
16S Ribosomal RNA V6 V4 Clin. Microbiol. Rev. 2003 V4 alimetrics.net Conserved region - unspecific Variable region – group or species specific
MiSeq is Flexible • PE250 and PE300 allows for flexibility V 6 V 4 when selecting target primers Can sequence barcodes of variable length • - 6, 8, 12nt possible Can sequence inline, single or dual • Conserved region - unspecific indices Variable region – group or species specific Manageable amount of Data for Lower Costs • 10-15 million reads per run $1300-1800 per run •
DNA Extraction
DNA Extraction Methods and Considerations What type of sample do I have? • Gut Contents/Fecal Soil/Environmental • Skin swabs • • BAL How much of each sample do I have? Hummingbird fecal material • Single skin swab • 1 Liter of wastewater • All the dirt you could carry •
Library Preparation
Adapter 2-TS_R 3’ 5’ Target Region 5’ 3’ Adapter 1-TS_F PCR-1 Product Target Region Extract target Adapter 1 TS_F TS_R Adapter 2 specific region P7-BC1-Adapter 2 Target Region Adapter 1 TS_R Adapter 2 TS_F P5-BC2-Adapter 1 PCR-2 Product Add sample Target Region specific barcodes P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7 and sequencing adapters Read 4 primer Read 4 (100-300bp) Sequencing Target Region P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7 Read 1 (100 - 300bp) Read 3 (8bp) Read 2 RC (8bp) Read 3 primer Read 2 RC primer Read 1 primer
Common Universal Primers 16S - Bacteria 27F and 1492R – Full Length (Sanger Sequencing) • • 27F and 534R – V1 to V3 319F and 806R – V3 to V4 • • 515F and 806R – V4 (Earth Microbiome Project) 515F and 926R – V4 to V5 (Earth Microbiome Project) • ITS – Internal Transcribed Spacer - Fungi ITS1f and ITS2 • • ITS3 and ITS4 ITS86F and ITS4 • 18S – Microbial Eukaryotes • Euk1391f and EukBr
Adapter 2-TS_R 3’ 5’ Target Region 5’ 3’ Adapter 1-TS_F PCR-1 Product Target Region Extract target Adapter 1 TS_F TS_R Adapter 2 specific region P7-BC1-Adapter 2 Target Region Adapter 1 TS_R Adapter 2 TS_F P5-BC2-Adapter 1 PCR-2 Product Add sample Target Region specific barcodes P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7 and sequencing adapters Read 4 primer Read 4 (100-300bp) Sequencing Target Region P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7 Read 1 (100 - 300bp) Read 3 (8bp) Read 2 RC (8bp) Read 3 primer Read 2 RC primer Read 1 primer
Benefits of Utilizing a Core Facility We navigate the pitfalls so you don’t have to Technology and methods rapidly evolve • Spend your time on your project instead of chasing best techniques • Leverage the Power of Scale We can combine your project with others to decrease costs • We validate all consumables and order larger volumes than a typical lab • Proven Expertise This is what we do day in, day out •
Host Microbe Systems Biology Core Thanks for Listening!!! For More Information contact: Matt Rolston mrrolston@ucdavis.edu Tupper Hall Room 3439 (530) 754-7850 http://www.ucdmc.ucdavis.edu/medmicro/hmsbcore/index.html
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