454 Sequencing System Guidelines for Amplicon Experimental Design June 2013 Instrument Kit GS Junior Junior GS FLX+ XL+ GS FLX+ XLR70 GS FLX XLR70 For life science research only. Not for use in diagnostic procedures. Guidelines for Amplicon Experimental Design 454 Sequencing System 454 Sequencing System Guidelines for Amplicon Experimental Design Table of Contents 1 General Guidelines for Amplicon Sequencing Experimental Design ...................... 5 1.1 Basic Considerations ................................................................................................................................. 6 1.2 Depth of Coverage ..................................................................................................................................... 6 1.3 Multiplexing ................................................................................................................................................. 7 1.3.1 Multiplex PCR ........................................................................................................................................................................ 7 1.3.2 Multiplex Identifiers (MIDs) ............................................................................................................................................. 7 1.4 Bidirectional vs. Unidirectional Sequencing ........................................................................................ 8 1.5 Quantitative Results in Amplicon Sequencing ..................................................................................... 8 2 Five Types of Amplicon Sequencing Experimental Design ........................................ 9 3 Design 1: Basic Amplicon Sequencing ........................................................................ 11 3.1 Library preparation................................................................................................................................... 12 3.1.1 Kit and Method .................................................................................................................................................................. 12 3.1.2 Multiplexing......................................................................................................................................................................... 13 3.1.3 Fusion Primer Design ...................................................................................................................................................... 14 3.1.4 Amplicon Length Considerations ............................................................................................................................... 15 3.1.5 Fusion Primer Ordering .................................................................................................................................................. 15 3.2 emPCR Amplification ............................................................................................................................... 16 3.2.1 Kits and Methods .............................................................................................................................................................. 16 3.2.2 Library Pooling ................................................................................................................................................................... 16 3.3 Sequencing ................................................................................................................................................ 17 3.3.1 Kit and Method .................................................................................................................................................................. 17 3.3.2 Pooling ................................................................................................................................................................................... 17 3.4 Data Processing........................................................................................................................................ 18 3.5 Data Analysis............................................................................................................................................. 18 4 Design 2: Universal Tailed Amplicon Sequencing ..................................................... 19 4.1 Library preparation................................................................................................................................... 20 4.1.1 Kit and Method .................................................................................................................................................................. 20 4.1.2 Multiplexing......................................................................................................................................................................... 21 4.1.3 Fusion Primer Design ...................................................................................................................................................... 22 4.1.3.1 Adding the Universal Tails ................................................................................................................................ 22 4.1.3.2 Targeting the Universal Tails ............................................................................................................................ 22 4.1.4 Amplicon Length Considerations ............................................................................................................................... 24 4.1.5 Fusion Primer Ordering .................................................................................................................................................. 24 June 2013 3 Guidelines for Amplicon Experimental Design 454 Sequencing System 4.2 emPCR Amplification ............................................................................................................................... 24 4.3 Sequencing ................................................................................................................................................ 24 4.4 Data Processing........................................................................................................................................ 24 4.5 Data Analysis............................................................................................................................................. 24 5 Design 3: Ligated Adaptors Amplicon Sequencing ................................................... 25 5.1 Library Preparation .................................................................................................................................. 26 5.2 emPCR Amplification ............................................................................................................................... 27 5.3 Sequencing ................................................................................................................................................ 27 5.4 Data Processing........................................................................................................................................ 27 5.5 Data Analysis............................................................................................................................................. 27 6 Design 4: Long Range PCR Amplicon Sequencing ................................................... 28 6.1 Library Preparation .................................................................................................................................. 29 6.2 emPCR Amplification ............................................................................................................................... 29 6.3 Sequencing ................................................................................................................................................ 29 6.4 Data Processing........................................................................................................................................ 29 6.5 Data Analysis............................................................................................................................................. 29 7 Design 5: One-Way Reads Amplicon Sequencing..................................................... 31 7.1 Library preparation................................................................................................................................... 33 7.1.1 Kit and Method .................................................................................................................................................................. 33 7.1.2 Multiplexing......................................................................................................................................................................... 34 7.1.3 Fusion Primer Design ...................................................................................................................................................... 34 7.1.4 Amplicon Length Considerations ............................................................................................................................... 35 7.1.5 Fusion Primer Ordering .................................................................................................................................................. 35 7.2 emPCR Amplification 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