PacBio RS II Worksflow

Transcript

1. FIND MEANING IN COMPLEXITY © Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved. PacBio® RS II Workflow
2. Overview of Release Agenda 2 PacBio RS II Instrument PacBio Software Sample Preparation PacBio® RS II Workflow Overview Where to find additional information
3. Sequencing Workflow Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis RS Remote RS Touch Blade Center SMRT® Portal & SMRT® View
4. Template Preparation DNA Sample Fragment DNA Damage Repair/ End Repair Ligate adapters Purify DNA SMRTbell™ Template preparation can be used to create libraries of various insert sizes from 250 bp to 20,000 bp depending on the needs of the application. Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
5. Run Design Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
6. Bind Polymerase to SMRTbell™ template Anneal Primer Bind Polymerase SMRTbell™ Template Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis Diffusion Loading MagBead Loading Two available methods for loading SMRTbell™ libraries
7. Instrument Run RS Touch Blade Center Reagents/Samples SMRT® Cell/ Tips Waste Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
8. Robot During SMRT® Cell Preparation Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
9. Primary Analysis Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
10.  Data Volume  Primary Analysis Movie-to-Trace Trace-to-Pulse Pulse-to-Base Circular Consensus GCAACGATCACCTAAA…GCAACGA TCACCTAAA…GCAACGATCACCTA AA…GCAACGATCACCTAAA… ACGATCACCTAAA…  Time ~4TB ~100 GB ~30 GB Algorithms reduce photon counts vs. time to sequences  ~ 30 min   > 60 min  Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis  log (size/GB) 
11. RS Dashboard Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
12. Secondary Analysis Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
13. SMRT® View Template Preparatio n Run Design Polymeras e Binding Instrument Run Primary Analysis Secondar y Analysis Tertiary Analysis Template Preparation Run Design Polymerase Binding Instrument Run Primary Analysis Secondary Analysis Tertiary Analysis
14. Where to Find More Information • Documents are available through the Customer Portal, SampleNet and DevNet – Template Prep − Guide - Pacific Biosciences® Template Preparation and Sequencing − Procedures & Checklists for 250 bp-10 kb libraries − PacBio guidelines – SMRTbell™ libraries – Software Getting Started Guide • Help Guides are available through the “Help” tab of the SW – RS Remote Guide (see next slide) – SMRT® Portal – SMRT® View • Help Guides and Informatics resources are also available in DevNet – SMRT® Portal Guide – SMRT® View Guide – Bioinformatic Training Wiki
15. RS Remote Help 15
16. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, and SMRTbell are trademarks of Pacific Biosciences in the United States and/or other countries. All other trademarks are the sole property of their respective owners.