Host Microbe Systems Biology Core Tupper Hall Room 3439 (530) - - PowerPoint PPT Presentation

Host Microbe Systems Biology Core

Tupper Hall Room 3439 (530) 754-7850 Director: Satya Dandekar, Ph.D. Associate Director: Loubna Tazi, Ph.D. Manager:

Matt Rolston

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 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) Because this gene has changed very little over time we can construct phylogenies to examine the evolution of bacteria and their relationships to

• ne another

The Beginning The End Tool for Generating New Hypotheses Tool for Generating the Final Answer

What 16S Sequencing IS and what it IS NOT

Powerful when combined with

• ther techniques like qPCR and

Gene Expression data A standalone technique that requires no other validation

16S sequencing is a valuable resource IF you understand its limitations

Qualitative with some quantitative elements depending on experimental design A purely quantitative tool that will solve all of your problems and wrap them up with a nice little bow IS IS NOT

Insect Models Agriculture Studies Mouse Models Primate Models Development Studies Nutrition Studies The Built Environment 16S Microbial Analysis Environmental Communities Human Health and Disease

Typical 16S Microbial Analysis

DNA Extraction

Qiagen (MoBio™) Purification Kits Amplification of 16S region Paired end barcoding

Library Construction

QIIME: Rob Knight, UC San Diego Mothur: Pat Schloss, Univ. of Michigan DADA2: BCallahan and SHolmes, Stanford

• 1. Sample Diversity
• 2. Community Structure
• 3. Conditional Modulations

Data Analysis Sequencing

Illumina MiSeq Potential output: 10 – 15 Gb

Workflow

UC Davis Host Microbe Systems Biology Core

Why the MiSeq for 16S?

Conserved region - unspecific Variable region – group or species specific

alimetrics.net

• Clin. Microbiol. Rev. 2003

16S Ribosomal RNA

V4 V6

V4

Conserved region - unspecific Variable region – group or species specific

MiSeq is Flexible

V 4 V 6

• PE250 and PE300 allows for flexibility

when selecting target primers

• Can sequence barcodes of variable length
• 6, 8, 12nt possible
• Can sequence inline, single or dual

indices

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 Adapter 1-TS_F

Target Region 5’ 5’ 3’ 3’ Target Region

Adapter 1 TS_F TS_R Adapter 2

PCR-1 Product Extract target specific region Target Region

P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7

Read 1 (100 - 300bp) Read 1 primer Read 4 (100-300bp) Read 4 primer Read 2 RC (8bp) Read 3 (8bp) Read 3 primer Read 2 RC primer Sequencing PCR-2 Product Add sample specific barcodes and sequencing adapters Target Region

P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7 P7-BC1-Adapter 2 P5-BC2-Adapter 1

Target Region

Adapter 1 TS_F TS_R Adapter 2

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

18S – Microbial Eukaryotes

• Euk1391f and EukBr
• ITS3 and ITS4
• ITS86F and ITS4

Target Region

Adapter 1-TS_F Adapter 2-TS_R

Target Region

Adapter 1 TS_F TS_R Adapter 2 P7-BC1-Adapter 2 P5-BC2-Adapter 1

PCR-1 Product Extract target specific region PCR-2 Product Add sample specific barcodes and sequencing adapters 5’ 5’ 3’ 3’ Target Region

Adapter 1 TS_F TS_R Adapter 2

Target Region

P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7

Target Region

P5 BC2 Adapter 1 TS_F TS_R Adapter 2 BC1 P7

Read 1 (100 - 300bp) Read 1 primer Read 4 (100-300bp) Read 4 primer Read 2 RC (8bp) Read 3 (8bp) Read 3 primer Read 2 RC primer Sequencing

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

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

Thanks for Listening!!!