BigDye™ Terminator v3.1 Cycle Sequencing Kit USER GUIDE Catalog Numbers 4337454, 4337455, 4337456, 4337457, 4337458 Publication Number 4337035 Revision C For Research Use Only. Not for use in diagnostic procedures.

The information in this guide is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history Table 1 Pub. No. 4337035 Revision Date Description C 14 April 2016 Updated compatible instrumentation, matrix and sequencing standards; added BigDye XTerminator™ purification and new basecalling reference sections. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Corporate entity Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288 TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. Centricon is a registered trademark of W. R. Grace and Co. Microsoft and Windows are registered trademarks of Microsoft Corporation. pGEM is a registered trademark of Promega Corporation. ©2016 Thermo Fisher Scientific Inc. All rights reserved.

Contents ■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Usage guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Compatible sequencing instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Thermal cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 ■ CHAPTER 2 Prepare templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Control template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Template preparation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 DNA isolation of single- and double-stranded templates . . . . . . . . . . . . . . . . . . . . . . . . 10 DNA isolation of BAC templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 DNA template amplification with PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Template quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Factors affecting template quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Determining template quality and quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Template quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 ■ CHAPTER 3 Perform cycle sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Set up the sequencing reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Using BigDye™ Terminator v1.1 \& v3.1 5X Sequencing Buffer to dilute sequencing reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Perform cycle sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Run the sequencing reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 ■ CHAPTER 4 Purify the sequencing reactions . . . . . . . . . . . . . . . . . . . . . . . . 16 Purify sequencing reactions with BigDye XTerminator™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Purify the sequencing reactions with Centri‑Sep™ plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Purify the sequencing reactions with ethanol/EDTA precipitation . . . . . . . . . . . . . . . . . . . . . 19 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 3

Contents ■ CHAPTER 5 Capillary electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Capillary electrophoresis guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Calibration guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Dye set and matrix standards for the 310 instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Dye set and spectral matrix standards for the 3130, 3500, and 3730 instruments . . . 22 Sequencing standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Electrophoresis on the 310 Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Performing electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Electrophoresis on the 3130/3130xl Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Performing electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Electrophoresis on the 3500/3500xL Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Performing electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Electrophoresis on the 3730/3730xl DNA Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Performing electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 ■ APPENDIX A Troubleshooting Sanger sequencing data . . . . . . . . . . . . . 26 Troubleshooting poor Sanger sequencing quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Recommended raw signal ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 The use of controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 No signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Example of no signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 No signal: possible causes and recommended actions . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Low signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Examples of low signal intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Low signal possible causes and recommended actions . . . . . . . . . . . . . . . . . . . . . . . . . 31 Mixed sequence throughout the electropherogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Example of mixed sequence throughout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Mixed sequence throughout possible causes and recommended actions . . . . . . . . . . 32 Mixed sequence up to or after a certain point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Example of mixed sequence after a point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Mixed sequence up to or after a certain point possible causes and recommended actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Poor mobility correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Example of poor mobility correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Poor mobility correction possible causes and recommended actions . . . . . . . . . . . . . 34 Dye blobs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Example of dye blobs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Dye blobs: possible causes and recommended actions . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Contents Signal saturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Examples of signal saturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Example of extreme signal saturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Signal saturation: possible causes and recommended actions . . . . . . . . . . . . . . . . . . . 38 Poor 5′ sequencing quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Example of poor 5′ sequencing quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Poor 5′ sequencing quality causes and actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 ■ APPENDIX B Select sequencing primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Primer considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Primer guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Optimize primer selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Primer Designer™ Tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 ■ APPENDIX C Partial sequence of pGEM™-3Zf(+) . . . . . . . . . . . . . . . . . . . . . 42 ■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 ■ APPENDIX E Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 5

1 Product information ■ Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 ■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 ■ Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 ■ Compatible sequencing instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 ■ Thermal cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 ■ Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix in this document. Product description The BigDye™ Terminator v3.1 Cycle Sequencing Kit provides pre-mixed reagents for Sanger sequencing reactions. The kit reagents are suitable for performing fluorescence-based cycle sequencing reactions on single-stranded or double-stranded DNA templates, on PCR fragments, and on large templates (for example, BAC clones). The kit includes BigDye™ Terminator v1.1 \& v3.1 5X Sequencing Buffer , which is specifically optimized for use with the BigDye™ Ready Reaction mixes. The kit has been formulated to deliver robust performance across a wide variety of DNA sequences while maximizing readlengths. When used in combination with Minor Variant Finder Software, the kit can also be used to detect variants as low as 5% in a sample (see Minor Variant Finder Software User Guide (Pub. No. MAN0014835). 6 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

1Chapter 1 Product information Workflow Workflow Kit contents and storage Table 2 BigDye™ Terminator v3.1 Cycle Sequencing Kit Contents Contents Cat. no. Cat. no. Cat. no. Cat. no. Cat. no. Storage 4337454 4337455 4337456 4337457 4337458 (24 reactions) (100 (1,000 (5,000 (25,000 reactions) reactions) reactions) reactions) BigDye™ Terminator 1 × 192 µL 1 × 800 µL 10 × 800 µL 2 × 20 mL 10 × 20 mL v3.1 Ready Reaction Mix 1 × 10 µL 1 × 250 µL 2 × 250 µL pGEM™-3Zf(+) double- 1 × 10 µL 1 × 10 µL 1 × 200 µL 2 × 200 µL Store at –15 stranded DNA Control 10 × 250 µL to –25°C. Template (200ng/ µL) 2 × 1 mL 1 × 12 mL 2 × 28 mL –21 M13 Control 1 × 10 µL 10 × 200 µL Primers (0.8pmol/ µL) BigDye™ Terminator 1 × 1 mL 10 × 28 mL Store at 4°C. v1.1 \& v3.1 5X Sequencing Buffer [1] [1] Shipped separately from the rest of the kit Usage guidelines • Avoid excess freeze-thaw cycles (no more than 10). If needed, aliquot reagents into smaller amounts. • Before each use of the kit, allow the frozen stocks to thaw on ice or at room temperature (do not heat). • Keep thawed materials on ice during use. Do not leave reagents at room temperature for extended periods. • Protect dyes from light to avoid photobleaching. IMPORTANT! Mix each stock thoroughly, then centrifuge briefly to collect all the liquid at the bottom of each tube. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 7

1 Chapter 1 Product information Compatible sequencing instruments Compatible sequencing instruments This guide provides general instructions for using the kit to generate samples for these instruments. For more detailed instructions, refer to the appropriate instrument or chemistry guide. • 310 Genetic Analyzer • 3130/3130xl Genetic Analyzer • 3500/3500xL Genetic Analyzer • 3730/3730xl DNA Analyzer Thermal cyclers The protocols provided in this guide were optimized using Applied Biosystems™ thermal cyclers, including the: • GeneAmp™ PCR System 9700 Dual 96-Well • GeneAmp™ PCR System 9700 Dual 384-Well • Veriti™ 96-Well Fast Thermal Cycler • Veriti™ 384-Well Thermal Cycler It is possible to use a different thermal cycler, although you may need to re-optimize the thermal cycling conditions. Ramping time is very important. If the thermal ramping time is too fast (>1°/second), poor (noisy) data may result. Required materials Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier. Item Source Reagents 4337456 10977015 BigDye™ Terminator v3.1 Cycle Sequencing Kit 4311320 or 4440753 UltraPure™ DNase/RNase-Free Distilled Water Hi‑Di™ Formamide Note: Not required for BigDye XTerminator™ Purification Kit purification. Sequencing and PCR primers (HPLC‑purified Primers can be designed and ordered with the Primer recommended) Designer™ Tool at http://www.thermofisher.com/ primerdesigner. See “Primer Designer™ Tool“ on page 41. Laboratory supplies MicroAmp™ Clear Adhesive Film 4306311 MicroAmp™ Optical 96-Well Reaction Plate 8010560 8 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

1Chapter 1 Product information Required materials Item Source Plate Septa, 96 well 4315933 MicroAmp™ Optical 384-Well Reaction Plate 4343370 Digital Vortex-Genie™ 2 or equivalent Scientific Inducstries, Inc. SI-A536 Centrifuge with swinging bucket (with PCR plate MLS adapter) Reagents for BigDye XTerminator™ Purification Kit purification (optional) BigDye XTerminator™ Purification Kit 4376486 Reagents for Centri‑Sep™ purification (optional) UltraPure™ SDS Solution, 10% 24730020 Centri‑Sep™ 96–Well Plates 4367819 Centri‑Sep™ 8–Well Strips (for <96 samples) 4367820 Reagents for ethanol/EDTA purification (optional) 0.5M EDTA, pH 8.0 for molecular biology AM9260G Ethanol, absolute, for molecular biology MLS BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 9

2 Prepare templates ■ Control template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 ■ Template preparation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 ■ Template quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 ■ Template quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Control template Include a control DNA template in every set of sequencing reactions. The results from the control help determine whether failed reactions are the result of poor template quality or sequencing reaction failure. • pGEM™-3Zf(+) is included with the kit as a double-stranded control (see Appendix C, “Partial sequence of pGEM™-3Zf(+)“ for the partial sequence) Note: The corresponding –21 M13 forward primer is also included. • M13mp18 is recommended as a single-stranded control. Template preparation methods Cycle Sequencing can be performed directly from single– or double–stranded DNA, plasmids, cosmids, BACs or purified PCR products. For high complexity DNA, PCR amplification of the target of interest before cycle sequencing is recommended. For general guidelines on DNA isolation, see DNA Sequencing by Capillary Electrophoresis Chemistry Guide (Pub. No. 4305080 or DNA Fragment Analysis by Capillary Electrophoresis User Guide (Pub. No. 4474504 for information on preparing single- and double-stranded templates. DNA isolation of For a comprehensive list of Thermo Fisher Scientific products available for DNA single- and isolation, go to https://www.thermofisher.com/us/en/home/life-science/ double-stranded dna-rna-purification-analysis/dna-extraction.html. templates DNA isolation of With larger DNA targets such as bacterial artificial chromosomes (BACs), DNA BAC templates template quality is important to the success of the sequencing reaction. Two methods have provided good sequencing results: • Alkaline lysis; include extra phenol extraction and isopropanol precipitation if very clean DNA is desired • Cesium chloride (CsCl) banding 10 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

2Chapter 2 Prepare templates Template quality For Thermo Fisher Scientific BAC DNA preparation products, go to: https://www.thermofisher.com/us/en/home/life-science/ dna-rna-purification-analysis/dna-extraction.html DNA template PCR templates can also be used to perform reliable cycle sequencing. For optimal amplification with results, purify PCR templates before sequencing. In general, any method that removes PCR unincorporated dNTPs and primers should work. See https://www.thermofisher.com/us/en/home/life-science/ dna-rna-purification-analysis/dna-extraction.html for a range of suitable kits. See the DNA Sequencing by Capillary Electrophoresis Chemistry Guide (Pub. No. 4305080 for information on sequencing PCR templates. Template quality DNA quality can significantly influence the length of the fragment that can be amplified and the reproducibility of amplification from one sample to another. Even if the fragment successfully amplifies, poor quality DNA can result in decreased signal or increased background fluorescent noise from the sequencing reactions. DNA quantity can also significantly effect amplification. For recommended quantities of DNA template per reaction, see “Template quantity“ on page 12. Factors affecting • Type and amount of source material – Influences the effectiveness and sensitivity template quality of PCR amplification and the quality of sequencing results. The number of sequencing targets relative to the amount of primer molecules can influence the efficiency and read-length of the sequencing reaction. • Contamination –  Can inhibit PCR amplification and cycle sequencing. Potential contaminants include: – Proteins, RNA or chromosomal DNA – Excess PCR primers, dNTPs, enzyme, and buffer components – Residual salts, organic chemicals such as phenol, chloroform, and ethanol, or detergents. – Heparin—can weaken or completely inhibit PCR amplification and cyclce sequencing. The Dynabeads™ DNA DIRECT™ Blood Kit and the QIAamp™ Blood Kit (QIAGEN, GmbH) successfully remove heparin from heparin blood samples, leaving genomic DNA ready for PCR amplification. Note: For parffin-embedded tissue, use a DNA isolation kit specifically designed formalin-fixed, paraffin-embedded (FFPE) tissue, and make sure that amplicon sizes are appropriate to the length of DNA fragment size that can be isolated. Smaller amplicons compatible with FFPE-fragmented DNA can be selected using the free Primer Designer™ Tool found at http://www.thermofisher.com/primerdesigner. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 11

2 Chapter 2 Prepare templates Template quantity Determining Use a spectrophotometer to determine DNA quality and to check for protein template quality contamination. Optimum absorbance ratios (A260/280) are between 1.8 and 2.0. and quantity If DNA and/or RNA contamination is suspected, run your sample on an agarose gel. A single band should be present for high quality DNA. For DNA quantitation, A260 values can be converted into µg/µL using Beer´s Law: • Concentration of single-stranded DNA = A260 × 33 mg/µL. • Concentration of double-stranded DNA = A260 × 50 mg/µL. Optical density (OD) measurements are used to determine template concentration. Highly concentrated (OD >1.0) or very dilute (OD <0.05) DNA samples can be inaccurate. Dilute or concentrate the DNA as needed to obtain an OD value between 0.05 to 1. Note: OD measurement is not a reliable method to determine template concentration following enzymatic PCR purification protocols. Instead, estimate PCR product purity and concentration using an agarose gel or a flourescence-based method like the PicoGreen™ reagent for use on the Qubit™ quantitation platform. Template quantity The table below lists the recommended quantity of template to use in a single cycle sequencing reaction. The quantity of PCR product is optimized to maximize the number of primer binding sites for the BigDye™ reaction and is dependent upon the length and purity of the PCR product. Note: In general, higher DNA quantities give higher signal intensities. Table 3 Recommended DNA quantities DNA template Quantity PCR product: 1–3 ng • 100–200 bp 3–10 ng • 200–500 bp 5–20 ng • 500–1000 bp 10–40 ng • 1000–2000 bp 20–50 ng • >2000 bp 25–50 ng 150–300 ng Single-stranded DNA 0.5–1.0 µg Double-stranded DNA 2–3 µg Cosmid, BAC Bacterial genomic DNA 12 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

3 Perform cycle sequencing ■ Prepare the reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 ■ Perform cycle sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Prepare the reactions Set up the IMPORTANT! Protect dye terminators from light. Cover the reaction mix and sequencing sequencing plates with aluminum foil before use. reactions 1. Completely thaw the contents of the BigDye™ Terminator v3.1 Cycle Sequencing Kit and your primers and store on ice. 2. Vortex the tubes for 2 to 3 seconds, then centrifuge briefly (2 to 3 seconds) with a benchtop microcentrifuge to collect contents at the bottom of the tubes. 3. Add components as indicated: IMPORTANT! Change pipette tips after each transfer. IMPORTANT! For control reactions, use 4 µL of the control primers for 20 µL and 10 µL reactions. Control primer concentration = 0.8pmol/µL. Standard reaction (20 µL) Standard reaction (10 µL) Component Quantity per Example Example Quantity per Example Example reaction Forward Reverse reaction Forward Reverse BigDye™ Terminator 3.1 Ready Reaction Mix 8 µL 8 µL 8 µL 4 µL 4 µL 4 µL Forward primer (3.2 µM) 3.2 pmol 2 µL — 3.2 pmol 1 µL — Reverse primer (3.2 µM) — 2 µL — 1 µL Deionized water Varies based on 8 µL 8 µL Varies based on 4 µL 4 µL (RNase/DNase-free) template and template and primer volume primer volume Template See “Template 2 µL[1],[2] 2 µL[1],[2] See “Template 1 µL[1], [2] 1 µL[1],[2] quantity“ on quantity“ on page 12 page 12 Total volume 20 µL 20 µL 20 µL 10 µL 10 µL 10 µL [1] e.g. 150-300ng/µL of dsDNA [2] Concentration of template may affect volume, if template volume differs, adjust the volume of water in the reaction mix. Note: Store on ice and protected from light. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 13

3 Chapter 3 Perform cycle sequencing Prepare the reactions 4. Seal the plate with MicroAmp™ Clear Adhesive Film. 5. Vortex the plate for 2 to 3 seconds, then centrifuge briefly in a swinging bucket centrifuge to collect contents to the bottom of the wells (5 to 10 seconds) at 1,000 x g. Note: Bubbles may be present within the wells, but do not adversely affect the reaction. Using BigDye™ Some cycle sequence reactions may be optimized using diluted BigDye™ Terminator Terminator v1.1 \& Ready Reaction Mix. The BigDye™ Terminator Ready Reaction Mix is provided at a v3.1 5X 2.5X concentration and can be diluted using BigDye™ Terminator v1.1 \& v3.1 5X Sequencing Buffer Sequencing Buffer to a final end reaction concentration of 1X. to dilute sequencing Calculate the volume of BigDye™ Terminator v1.1 \& v3.1 5X Sequencing Buffer to use: reactions 0.5 * ((total reaction volume)/2.5) - volume of BigDye™ Terminator Ready Reaction Mix) Note: Dilution of the BigDye™ Terminator v1.1 \& v3.1 5X Sequencing Buffer without optimization, may cause deterioration of sequencing quality. We can not guarantee the performance of BigDye™ chemistry when it is diluted. Component Diluted reaction (0.5X) Quantity per reaction Example Forward Example Reverse BigDye™ Terminator 3.1 Ready Reaction Mix BigDye™ Terminator v1.1 \& v3.1 5X Sequencing 4 µL 4 µL 4 µL Buffer 2 µL 2 µL 2 µL Forward primer (3.2 µM) 3.2 pmol 2 µL[1] — Reverse primer (3.2 µM) — 2 µL[1] Deionized water (RNase/DNase-free) Varies based on 10 µL 10 µL template and primer volume Template See “Template 2 µL[2],[3] 2 µL[2],[3] quantity“ on page 12 Total volume 20 µL 20 µL 20 µL [1] The control primer is provided at 0.8pmol/µL. Use 8 µL to obtain a total primer quantity of 3.2 pmol per 20 µL reaction. If primer volume differs, adjust the volume of water in the reaction mix. [2] e.g. 150-300ng/µL of dsDNA [3] Concentration of template may affect volume, if template volume differs please adjust the volume of water in the reaction mix. 14 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

3Chapter 3 Perform cycle sequencing Perform cycle sequencing Perform cycle sequencing Run the 1. Place the tubes or plate(s) in a thermal cycler and set the correct volume: sequencing reactions • 20 µL for microcentrifuge tubes or 96-well reaction plates • 10 µL for 384-well reaction plates 2. Perform cycle sequencing: Stage/step Parameter Incubate 25 cycles Hold Ramp rate — Denature Anneal Extend 4°C Temperature 96°C Hold until ready Time (mm:ss) 01:00 96°C 1°C/second to purify. 00:10 50°C 60°C 00:05 04:00[1] [1] Shorter extension times can be used for short templates. 3. Briefly centrifuge the reactions and proceed to Chapter 4, “Purify the sequencing reactions“. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 15

4 Purify the sequencing reactions ■ Purify sequencing reactions with BigDye XTerminator™ . . . . . . . . . . . . . . . . . . . 16 ■ Purify the sequencing reactions with Centri-Sep™ plates . . . . . . . . . . . . . . . . . . 17 ■ Purify the sequencing reactions with ethanol/EDTA precipitation . . . . . . . . . . . 19 Salts, unincorporated dye terminators, and dNTPs in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling. Three different methods to purify sequencing products are provided below. For a list of the required materials for each method, see “Required materials“ on page 8. Purify sequencing reactions with BigDye XTerminator™ The following protocol takes approximately 40 minutes. For detailed instructions, see the BigDye XTerminator™ Purification Kit Protocol (Pub. No. 4374408). Note: Use disposable reagent reservoirs and an 8-channel P200 pipette, if available, to facilitate the clean-up process. Use wide bore pipet tips when pipetting the BigDye XTerminator™ reagent. Note: If you use a 3730 DNA Analyzer, standard heat sealing techniques can be used. This protocol describes plate sealing with MicroAmp™ Clear Adhesive Film. The MicroAmp™ Clear Adhesive Film must be removed before loading the plate on the instrument. 1. Vortex the bottle of BigDye XTerminator™ beads for 8 to 10 seconds before mixing with the SAM solution. IMPORTANT! For effective BigDye XTerminator™ clean-up, it is essential to keep the materials well mixed. Keep reagents on ice between pipetting steps. 2. Prepare the SAM/BigDye XTerminator™ bead working solution: Component Volume per 10 µL reaction Volume per 20 µL reaction SAM solution 45 µL 90 µL BigDye XTerminator™ bead solution 10 µL 20 µL Total volume 55 µL 110 µL 3. Remove MicroAmp™ Clear Adhesive Film from the sequencing plate. 16 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Chapter 4 Purify the sequencing reactions 4 Purify the sequencing reactions with Centri‑Sep™ plates 4. Transfer the indicated volume of bead mix (BigDye XTerminator™ bead solution and SAM solution) to each sample. IMPORTANT! To mix thoroughly, aspirate and dispense the solution 3 to 4 times before each transfer. Re-mix solution after each dispense step. 5. Seal the plate using MicroAmp™ Clear Adhesive Film. 6. If you are using the Digital Vortex-Genie™ 2, vortex the 96-well plate for 20 minutes at 1,800 rpm. For alternative vortex mixer manufacturers and settings, see the BigDye XTerminator™ Purification Kit Quick Reference Card (Pub. No. 4383427). 7. In a swinging bucket centrifuge, centrifuge the plate at 1,000 x g for 2 minutes. Note: To store for up to 10 days, seal the plate with MicroAmp™ Clear Adhesive Film, and store at 4°C for capillary electrophoresis (CE) preparation or at –20°C until use. BDX plates can be stored at room temperature for up to 48 hours inclusive of time on the CE instrument. Purify the sequencing reactions with Centri‑Sep™ plates The following protocol takes approximately 45 minutes (~25 minutes for purification and ~20 minutes for drying). IMPORTANT! Do NOT skip the drying step in this procedure. Running samples that have not been dried can affect signal variation when injected on CE instrumentation due to nature of the highly purified/desalted elution solution. Note: Individual Centri-Sep™ Spin columns can be used if few sequencing reactions need to be purified. Centri-Sep™ Spin columns must be hydrated for approximately 2 hours before use. See the DNA Sequencing by Capillary Electrophoresis Chemistry Guide (Pub. No. 4305080) for more information. 1. Prepare 2.2% SDS (sodium dodecyl sulfate) in standard deionized water. Note: Store 2.2% SDS at room temperature. The SDS will precipitate at 4°C or below. 2. Briefly centrifuge the sequencing plate in a swinging bucket centrifuge (5 to 10 seconds) at 1,000 x g. 3. Remove the MicroAmp™ Clear Adhesive Film. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 17

4 Chapter 4 Purify the sequencing reactions Purify the sequencing reactions with Centri‑Sep™ plates 4. Prepare the SDS heat treatment: Volume Component 10 µL 20 µL Sequencing reaction 10 µL — UltraPure™ DNase/RNase-Free Distilled Water 2 µL 2 µL 2.2% SDS 22 µL 22 µL Total volume 5. Vortex the plate for 2 to 3 seconds, then centrifuge briefly (5 to 10 seconds) at 1,000 x g. 6. Perform the SDS heat treatment. Parameter Denature Stage/step Hold 98°C Incubate 4°C Temperature 5 min 25°C Hold Time 10 min 7. Prepare the Centri-Sep™ 96-well plate: Note: The Centri-Sep™ 96-well plates come pre-hydrated. The initial centrifugation step removes the hydration solution. a. Allow the plate to equilibrate to room temperature. b. Place the Centri-Sep™ 96-well plate in an empty 96-well plate. c. Centrifuge for 2 minutes at 1,500 x g to remove the hydration solution from the plate. d. Discard the plate with flow-through hydration solution. e. Place a new MicroAmp™ Optical 96-Well Reaction Plate beneath the prepared Centri-Sep™ 96-well plate to collect purified BigDye™ sequencing reaction product. 8. Briefly centrifuge the SDS heat-treated extension product plate in a swinging bucket centrifuge (5 to 10 seconds) at 1,000 x g and remove the MicroAmp™ Clear Adhesive Film. 9. Dispense 20 µL SDS heat-treated extension product to the corresponding Centri-Sep™ well. Dispense slowly into the center of the well (e.g. electronic pipette setting 4). Do not touch the sides of the well or the gel material. 10. Place a new 96-well collection plate beneath the Centri-Sep™ plate. Using a swinging bucket centrifuge, centrifuge the Centri-Sep™ plate containing the SDS heat treated sample for 2 minutes at 1,500 x g to collect purified sample. 18 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

4Chapter 4 Purify the sequencing reactions Purify the sequencing reactions with ethanol/EDTA precipitation 11. Dry the sample in a vacuum centrifuge without heat or on the low heat setting for 10 to 15 minutes or until dry. Note: To store, seal the plate with MicroAmp™ Clear Adhesive Film, and store at 4°C for CE preparation or –20°C until use. Purify the sequencing reactions with ethanol/EDTA precipitation The following protocol takes approximately 90 minutes. Note: This method produces a clean signal, but it can cause subtle loss of small molecular weight fragments. IMPORTANT! Absolute ethanol absorbs water from the atmosphere, which gradually decreases its concentration and can affect sequencing results. Store appropriately and replace frequently. 1. Prepare a 125 mM EDTA solution from 0.5 M EDTA, pH 8.0. 2. Prepare 70% ethanol using absolute ethanol. Note: Replace every 2 weeks. IMPORTANT! Do NOT pre-mix 125 mM EDTA solution and absolute ethanol. This can cause precipitation of the EDTA. 3. Briefly centrifuge the sequencing plate in a swinging bucket centrifuge (5 to 10 seconds) at 1,000 x g. 4. Remove the MicroAmp™ Clear Adhesive Film from the plate. 5. Add the following in order: Volume Component 10 µL 20 µL sequencing reaction (starting volume) 2.5 µL 5 µL 125 mM EDTA solution 30 µL 60 µL absolute ethanol 42.5 µL/ well 85 µL/ well Total volume IMPORTANT! Dispense EDTA directly into the sample in each well. If droplets are visible on the wall of the well, briefly centrifuge the plate to ensure that the EDTA mixes with the sequencing reactions. 6. Seal the plate with MicroAmp™ Clear Adhesive Film. 7. Vortex the plate for 2 to 3 seconds, then centrifuge briefly (5 to 10 seconds) at 1,000 x g. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 19

4 Chapter 4 Purify the sequencing reactions Purify the sequencing reactions with ethanol/EDTA precipitation 8. Incubate the plate at room temperature for 15 minutes. IMPORTANT! Timing of this step is critical. 9. Centrifuge the plate in a swinging bucket centrifuge at 1,870 x g (4°C) for 45 minutes. IMPORTANT! Proceed to the next step immediately. If this is not possible, then centrifuge the plate for 2 minutes before performing the next step. 10. Slowly remove the MicroAmp™ Clear Adhesive Film to prevent disruption of the pellet. Place 4 layers of absorbent paper into the centrifuge and carefully invert the plate onto the paper without dislodging the pellet. Centrifuge at 185 × g for 1 minute. Do not tip out liquid first. Do not tap plate to facilitate liquid removal. 11. Add 70% ethanol to each well. Volume 70% ethanol Starting reaction volume 30 µL 10 µL 60 µL 20 µL 12. Seal the plate with MicroAmp™ Clear Adhesive Film and centrifuge at 1,870 × g (4°C) for 15 minutes. IMPORTANT! Proceed to the next step immediately. If this is not possible, then centrifuge the plate for 2 minutes before performing the next step. 13. Slowly remove the MicroAmp™ Clear Adhesive Film to prevent disruption of the pellet. Place 4 layers of absorbent paper into the centrifuge and carefully invert the plate onto the paper towel without dislodging the pellet. Centrifuge at 185 × g for 1 minute. Note: Do not tip out liquid first. Do not tap plate to facilitate liquid removal. 14. Allow the plate to air dry, face up and protected from light, for 5 to 10 minutes at room temperature. Note: To store, seal the plate with MicroAmp™ Clear Adhesive Film, and store, protected from light, at 4°C for CE preparation or –20°C until use. 20 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

5 Capillary electrophoresis ■ Capillary electrophoresis guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 ■ Calibration guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 ■ Electrophoresis on the 310 Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ■ Electrophoresis on the 3130/3130xl Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . 23 ■ Electrophoresis on the 3500/3500xL Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . 24 ■ Electrophoresis on the 3730/3730xl DNA Analyzer . . . . . . . . . . . . . . . . . . . . . . . . 24 Capillary electrophoresis guidelines We recommend that you verify the quality of your current matrix file or spectral calibration before proceeding. To generate a new matrix file or spectral calibration, use the appropriate matrix and/or sequencing standard for your instrument. The existing mobility files can be used with their respective platforms. • Resuspend purified and dried sequencing reactions in 10-µL of Hi-Di™ Formamide Do not heat samples to resuspend. Run samples as soon as possible after resuspension. Note: It is not necessary to resuspend samples purified with the BigDye XTerminator™ Purification Kit. • Standard heat seal consumables can only be used for the 3730/3730xl DNA Analyzer. • Select the correct mobility file for your instrument, polymer and dye chemistry. For example: Select KB_3500_POP7_BDTv3.mob for data electrophoresed on a 3500/3500xL Genetic Analyzer with POP-7™ polymer and generated with BigDye™ Terminator v3.1 chemistry. • Use the BDx run modules if you used the BigDye XTerminator™ Purification Kit for sequencing reaction clean up. • If the wrong mobility file is used, this can be corrected with Sequencing Analysis Software. For more information go to DNA Sequencing Analysis Software 6 (Pub. No. 4474239). • Check At PCR Stop in the Basecalling tab and the Use Mixed Base Identification in the Mixed Bases tab when analyzing PCR products with Sequencing Analysis Software. Do not select these settings when performing de novo or plasmid sequencing. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 21

5 Chapter 5 Capillary electrophoresis Calibration guidelines Calibration guidelines Matrix or sequencing standards provide a sample for multi-color spectral correction for the dye emission overlap of the BigDye™ Terminators. Spectral calibrations for the BigDye™ Terminator v1.1 are not compatible with BigDye™ Terminator v3.1. Perform new spectral calibrations after you install a capillary array or move capillaries in the detection areas. See your specific instrument user guide for more information on calibration. Dye set and matrix Instrument Dye set Standards for instrument (matrix) file standards for the generation [1] 310 instrument 310 Genetic Analyzer E 310 Genetic Analyzer Matrix Standards, BigDye™ Terminator v3.1 (Cat. No. 4336948) [1] Refer to the matrix or sequence standards product insert for instructions on generating matrices. Dye set and Instrument Dye set Standards for spectral calibration [1] spectral matrix Z standards for the 3130/3130xl Genetic 31xx Matrix Standards Kit, BigDye™ 3130, 3500, and Analyzer Terminator v3.1 (Cat. No. 4336974 ) 3730 instruments 3500/3500xL Genetic Analyzer 3730 DNA Analyzer N/A[2] [1] Refer to the matrix or sequence standards product insert for instructions on performing spectral calibrations. [2] Matrix standards are not designed for use on the 3730 DNA Analyzer; use sequencing standards only. Sequencing Instrument Kit Cat. no. standards 4336935 310 Genetic Analyzer 310/31xx Genetic Analyzer 3130/3130xl Genetic Sequencing Standards, 4404312 Analyzer BigDye™ Terminator v3.1 4336943 3500/3500xL Genetic Analyzer 3500/3500xL Sequencing Standards, BigDye™ 3730/3730xl DNA Analyzer Terminator v3.1 3730/3730xl DNA Analyzer Sequencing Standards, BigDye™ Terminator v3.1 22 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

5Chapter 5 Capillary electrophoresis Electrophoresis on the 310 Genetic Analyzer Electrophoresis on the 310 Genetic Analyzer Item Module name Polymer Capillary length Mobility files P4StdSeq (1 mL) E POP-4™ Dye set E P4RapidSeq (1 mL) E POP-4™ 36‑cm KB_310_POP4_BDTv3_36Std.mob run Seq POP6 Rapid (1 mL) E POP-6™ modules Seq POP6 (1 mL) E POP-6™ 36‑cm KB_310_POP4_BDTv3_36Rapid.mob 36cm KB_310_POP6_BDTv3_36Rapid.mob 50‑cm KB_310_POP6_BDTv3_50Std.mob Performing For information on performing sample electrophoresis on the 310 instrument, see the electrophoresis 310 Genetic Analyzer Manual for Windows™ (Pub. No. 4317588). Electrophoresis on the 3130/3130xl Genetic Analyzer Item Module name Polymer Capillary length Mobility files POP-4™ Dye set Z run UltraSeq36_POP4 POP-4™ 36-cm KB_3130_POP4_BDTv3.mob POP-4™ modules StdSeq50_POP4 POP-6™ 50-cm POP-6™ LongSeq80_POP4 POP-7™ 80-cm POP-7™ RapidSeq36_POP6 POP-7™ 36-cm KB_3130_POP6_BDTv3.mob POP-7™ StdSeq50_POP6 POP-7™ 50-cm UltraSeq36_POP7 36-cm KB_3130_POP7_BDTv3.mob RapidSeq36_POP7 36-cm FastSeq50_POP7 50-cm StdSeq50_POP7 50-cm LongSeq80_POP7 80-cm Performing For information on performing sample electrophoresis on the 3130/3130xl Genetic electrophoresis Analyzer, see: • Applied Biosystems™ 3130/3130xl Genetic Analyzers Getting Started Guide (Pub. No. 4477796) • Applied Biosystems™ 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and Reference Guide (Pub. No. 4352716 ) BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 23

5 Chapter 5 Capillary electrophoresis Electrophoresis on the 3500/3500xL Genetic Analyzer Electrophoresis on the 3500/3500xL Genetic Analyzer Item Module name Polymer Capillary length Mobility files POP-6™ Dye set Z run StdSeq50_POP6 POP-6™ 50-cm KB_3500_POP6_BDTv3.mob POP-6™ modules RapidSeq36_POP6 POP-6™ 36-cm POP-7™ RapidSeq50_POP6 POP-7™ 50-cm POP-7™ FastSeq50_POP6 POP-7™ 50-cm POP-7™ StdSeq50_POP7 POP-7™ 50-cm KB_3500_POP7_BDTv3.mob RapidSeq36_POP7 36-cm RapidSeq50_POP7 50-cm FastSeq50_POP7 50-cm ShortReadSeq50_POP7 50-cm MicroSeq_POP7 50-cm Performing For information on performing sample electrophoresis on the 3500/3500xL Genetic electrophoresis Analyzer, see: • 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3.1 User Guide (Pub. No. 100031809 ) • 3500 Series Genetic Analyzer Software Compatibility Matrix and User Documents Reference (Pub. No. 4485219 ) Electrophoresis on the 3730/3730xl DNA Analyzer Item Module name Polymer Capillary length Mobility files StdSeq36_POP-6™ POP-6™ Dye set Z LongSeq50_POP-6™ POP-6™ 36-cm KB_3730_POP6_BDTv3.mob run StdSeq36_POP-7™ POP-7™ modules FastSeq50_POP-7™ POP-7™ 50-cm RapidSeq36_POP-7™ POP-7™ TargetSeq36_POP-7™ POP-7™ 36-cm KB_3730_POP7_BDTv3.mob LongSeq50_POP-7™ POP-7™ XLRSeq50_POP-7™ POP-7™ 50-cm 36-cm 36-cm 50-cm 50-cm 24 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Performing 5Chapter 5 Capillary electrophoresis electrophoresis Electrophoresis on the 3730/3730xl DNA Analyzer For information on performing sample electrophoresis on the 3730/3730xl DNA Analyzer, see: • 3730/3730xl DNA Analyzer Software Compatibility Matrix and Reference Documents (Pub. No. 4449681) • Chemistry Guide, 3730/3730xl DNA Analyzer (Pub. No. 4331467) • User Guide: Applied Biosystems™ 3730/3730xl DNA Analyzer (Pub. No. 4331468) • Applied Biosystems™ 3730/3730xl DNA Analyzers Maintenance, Troubleshooting, and Reference Guide (Pub. No. 4359473) BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 25

A Troubleshooting Sanger sequencing data Troubleshooting poor Sanger sequencing quality Common sources of noise How to recognize the source Page Number 28 No signal Little to no raw signal. 30 Low signal intensity The raw signal is below the recommended minimum relative 32 fluorescence units (RFU). 33 Mixed sequence throughout Mixed sequence content throughout the length of the trace. 34 35 Mixed sequence up to or Mixed sequence content starting at a specific point. 37 after a point 39 Poor mobility correction Peaks overlapped and unevenly spaced Dye blobs Large broad peak normally seen at 85–90 bp or 125–130 bp. Signal saturation The raw signal exceeds the recommended maximum RFU. Note: Excessive raw signal causes pull-up peaks in the analyzed data, which can incorrectly be identified as mixed bases. Poor 5′ sequencing quality Poor quality sequence in the first 35 nucleotides of the trace. For more information on Sanger sequencing symptoms and troubleshooting, see the DNA Sequencing by Capillary Electrophoresis Chemistry Guide (Pub. No. 4305080). 26 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

AAppendix A Troubleshooting Sanger sequencing data Recommended raw signal ranges Recommended raw signal ranges Different instruments have different recommended fluorescence ranges and levels at which signal is saturated. Data quality can be compromised when signals do not fall in the appropriate range. The subsequent troubleshooting sections will help you to recognize and correct issues relating to raw fluorescent signals. Instrument Recommended raw signal range in relative Fluorescence saturation fluorescent units (RFU) 310 150– 4,000 8,100 3130/3130xl 150–4,000 8,100 3500/3500xl 150–10,000 32,000 3730/3730xl 175–10,000 32,000 The use of controls The use of controls are recommended to simplify troubleshooting. • Control DNA template (pGEM)—Results can help you determine whether failed reactions are caused by poor template quality or sequencing reaction failure. See “Control template“ on page 10. • Big Dye Terminator Sequencing standards—Results can help you distinguish between chemistry problems and instrument problems. • Use of Hi-Di Formamide only injection (Optional)—Results can help distinguish sample problems and instrument problems related to contamination. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 27

A Appendix A Troubleshooting Sanger sequencing data No signal No signal Lack of signal can be determined by looking at the scale of signal produced in the raw data view. Signal should be above the minimum recommended RFU. See Example of no “Recommended raw signal ranges“ on page 27. Lack of signal can be caused by many signal factors. These include problems in the sequencing reaction (template quantity/quality), thermal cycler malfunction (plate failure) and capillary electrophoresis failure (failing laser, air bubbles in lines, etc.). No signal due to hardware failure or a failed reaction No signal: possible causes and recommended actions Possible cause Recommended action Insufficient template Quantitate the DNA template. Inhibitory contaminant in the template Increase the amount of DNA in the sequencing reactions. See “Template quantity“ on page 12. Clean up the template. See Chapter 2, “Prepare templates“ Insufficient primer Quantitate the primer and increase the amount of primer in the sequencing reactions if needed. 28 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Possible cause AAppendix A Troubleshooting Sanger sequencing data Primer has no annealing site No signal Poor primer design or incorrect primer sequence Missing reagent Recommended action Old or mishandled reagents Thermal cycler malfunction Use a primer that is complementary to the template. Thermal cycling conditions incorrect Review the primer design and if needed redesign the Extension products lost during reaction cleanup primer. Extension products not resuspended Repeat the reactions, carefully following the protocol. Electrokinetic injection failure Use pGEM control to confirm reagent workflow perfromance. Use fresh reagents. Test the thermal cycler per the manufacturer's instruction and repeat the reactions. Calibrate the thermal cycler regularly. Use the correct thermal cycling parameters. Use the correct tubes or plates for your thermal cycler. Set the ramp rate to 1°C/second. Use the correct centrifuge speeds and times for the precipitation procedures and the spin column or spin plate procedures. Check that the ethanol concentration is correct for the precipitation protocols. Carefully resuspend the sample pellet in Hi‑Di™ Formamide. Run the Sequencing standard to confirm the performance of the instrument and capillary array. Confirm the BDX run module is being used for BigDye XTerminator™‑purified samples. Confirm the correct volume is in the well. Confirm that the sample plate does not have a bubble at the bottom of the well. Briefly centrifuge to remove bubble. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 29

A Appendix A Troubleshooting Sanger sequencing data Low signal Low signal Low signal intensity can be caused by many factors including thermal cycler malfunction (in the case of an entire plate failure) and insufficient sequencing Examples of low template quantity/quality. signal intensity The examples below show severely low signal traces. Figure 1 Severely low signal intensity Figure 2 Severely low signal intensity due to hardware failure or a failed reaction More severe signal issues are often related to poor injection, failed reaction, or a blocked or broken capillary. 30 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

AAppendix A Troubleshooting Sanger sequencing data Low signal Low signal Note: When sequencing signal is weak, increasing the injection time (re-injecting possible causes sample) or increasing primer and/or template in the cycle sequencing reactions can and recommended improve signal strength if DNA quality, PCR purification, and sequencing reaction actions purification steps have been performed properly. Possible cause Recommended action Insufficient DNA in the sequencing reactions Increase DNA quantity in the sequencing reactions. See “Template quantity“ on page 12. Load or inject more of the resuspended sequencing reactions by modifying the run module and increasing the injection time. See the appropriate instrument User Guide for additional information.. Poor primer quality or quantity Prepare a fresh working stock of primer or order new primer. Use 3.2pmol in the final reaction. Degraded template Prepare fresh DNA and repeat the reactions. Poor PCR cleanup Purify PCR products before use. See “DNA template amplification with PCR“ on page 11. Old or mishandled reagents Use fresh reagents. Thermal cycling conditions incorrect Calibrate the thermal cycler regularly. Use the correct thermal cycling parameters. Use the correct tubes or plates for your thermal cycler. Set the ramp rate to 1°C/second. Insufficient Ready Reaction Mix in the reactions Sequencing chemistry was diluted. See “Prepare the reactions“ on page 13 for recommended procedures. Note: Thermo Fisher Scientific can not support diluted reactions or guarantee the performance of diluted BigDye chemistry. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 31

A Appendix A Troubleshooting Sanger sequencing data Mixed sequence throughout the electropherogram Mixed sequence throughout the electropherogram Example of mixed sequence throughout Figure 3 Secondary sequence contamination caused by well‑to‑well contamination of one sample into another Mixed sequence throughout possible causes and recommended actions Possible cause Recommended action Inhibitory contaminant in the template Multiple templates in the sequencing reaction Clean up the template. Multiple priming sites Examine the template on an agarose gel to be sure that Multiple primers when sequencing PCR products only one template is present. Primer with N‑1 contamination High signal saturating the detector Verify that the primer has only one priming site. Incorrect run module If needed, redesign the primer. See Appendix B, “Select Incorrect instrument (matrix) file sequencing primers“ Purify the PCR template to remove excess primers. Use an HPLC‑purified primer Use less DNA in the sequencing reactions. See “Template quantity“ on page 12. Load or inject less of the resuspended sequencing reactions by modifying the run module and decreasing the injection time. See the appropriate instrument User Guide for additional information. Use a default run module. Use the correct instrument file for BigDye™ Terminator chemistry. 32 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

AAppendix A Troubleshooting Sanger sequencing data Mixed sequence up to or after a certain point Mixed sequence up to or after a certain point Example of mixed sequence after a point Figure 4 Example of mixed sequence content following a heterozygous insertion or deletion. Mixed sequence content is seen in both forward and reverse traces. Mixed sequence up to or after a certain point possible causes and recommended actions Possible cause Recommended action Mixed plasmid separation Be sure that you have only one template. Multiple PCR products Primer-dimer contamination in PCR sequencing Optimize your PCR amplification. Be sure that there is no sequence complementarity Slippage after repeat region in template between the two PCR primers. Be sure that your sequencing primer does not overlap the sequences of the PCR primers. Use a Hot Start technique, such as with AmpliTaq Gold™ DNA Polymerase. Try an alternative sequencing chemistry. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 33

A Appendix A Troubleshooting Sanger sequencing data Poor mobility correction Possible cause Recommended action Slippage after repeat region in template Use an anchored primer. Heterozygous insertion or deletion mutation (HIM) Some customers have gotten past poly(A) regions using a mixture of oligo dT18 primers with either a C, A, or G as the 3′ terminal dinucleotide or 2-base anchors. See the DNA Sequencing by Capillary Electrophoresis Chemistry Guide (Pub. No. 4305080) for more information. Obtain forward and reverse sequence data and assemble using SeqScape™ Software or Variant Reporter™ Software. • SeqScape™ Software lists HIMs in the Mutations Report. Clicking the Base Change in the Mutations Report to view the mutation in the Project view. • Variant Reporter™ Software lists HIMs in the Project Summary Report. Poor mobility correction Example of poor mobility correction Example electropherogram with the wrong mobility file selected. Poor mobility correction possible causes and recommended actions Possible cause Recommended action Incorrect dye set/primer (mobility) file Use the correct dye set/primer file. Incorrect Peak 1 location for data analysis Select a new Peak 1 location. 34 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Dye blobs AAppendix A Troubleshooting Sanger sequencing data Dye blobs Example of dye blobs Dye blobs are caused by unincorporated dye terminators remaining in solution after purification of the cycle sequencing reactions. Unincorporated dye terminators from the BigDye™ Terminator v3.1 Cycle Sequencing Kit and BigDye™ Direct Cycle Sequencing Kit are most commonly seen to co-migrate with the ~ 85–90 bp labeled fragments. In more severe instances, these blobs can also be detected at ~ 60–65 bp and within 125–140 bp regions. Dye blobs are typically seen as broad “C” or “T” peaks, but can also show up as “G” blobs. Dye blobs are more common when first testing new sequencing purification methods. Figure 5 shows severe dye blobs in the 60–65bp, 85–100bp, and 125–140bp regions. Although the sequence quality appears high, the blobs obscure nearly 40 bp of the 100 bases displayed. This would make the sequence unsuitable for variant detection. Figure 5 Severe dye blobs in the 60–65bp and 125–140bp regions Dye blobs: possible causes and recommended actions Possible cause Recommended action Sample bypassed the purification material when using Ensure transfer of the sample to the center of the spin columns/spin plates for sequencing clean‑up. purification material without the pipet tip touching the purification material. Sample dispensed along the walls of the clean‑up column may bypass the purification material. Use a single channel pipette and/or position the tip directly above the spin column/plate while dispensing at low speed. Ethanol concentration is too high during ethanol Repeat procedure with correct ethanol concentration. precipitation. This leads to unincorporated dye terminators and salts precipitating with the sequencing product. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 35

A Appendix A Troubleshooting Sanger sequencing data Dye blobs Possible cause Recommended action Incorrect ratio of BigDye XTerminator™ reagents. Vortex theBigDye XTerminator™ Solution bulk container at maximum speed for at least 10 seconds before dispensing. Use wide bore tips when pipetting Xterminator solution. If you pre‑mix the SAM/BDX solution, ensure that the solution is well mixed before each sample well dispense step to maintain the appropriate ratio of reagents. Insufficient mixing during the vortexing step when using Verify that the plate is firmly attached to the vortexer. the BigDye XTerminator™ Purification Kit. Follow the protocol for vortexing. See BigDye XTerminator™ Purification Kit Quick Reference Card (Pub. No. 4383427) for recommended vortexers. 36 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

AAppendix A Troubleshooting Sanger sequencing data Signal saturation Signal saturation High sample signal causes saturation of the CCD camera. Signal saturation causes pull-up spectral peaks that cannot be corrected by spectral calibration. Extreme signal saturation will appear as mixed sequence. Note: The 3500 Data collection software flags .ab1 files with off-scale peaks. You must manually check for off-scale peaks from data generated with the 310 Genetic Analyzer, the 3130/3130xl Genetic Analyzer or the 3730/3730xl DNA Analyzer platforms. Examples of The following figure shows examples of signal saturation. The red line indicates the signal saturation maximum raw signal recommended. 1 2 3 Figure 6 Signal saturation – Raw data view 3 Minor signal saturation on a 3500 Genetic Analyzer 1 Severe signal saturation on a 3130 Genetic Analyzer 2 Minor signal saturation on a 3130 Genetic Analyzer BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 37

A Appendix A Troubleshooting Sanger sequencing data Signal saturation Example of extreme signal saturation Mixed sequence in the Analyzed view. Mixed sequence can be due to extreme signal saturation caused by pull-up peaks. A quick review of the raw data can help diagnose a scenario such as this; the raw data view of the analyzed sample shown here is shown in the top panel in Figure 6. Signal saturation: possible causes and recommended actions Possible cause Recommended action Too much template was used in the sequencing reaction If the sample has been on instrument <24 hours, reduce resulting in too much sequencing product. injection time in run module, then re‑inject the sample. If the sample is purified with the BigDye XTerminator™ Purification Kit and has been on instrument <24 hours, carefully remove 10 µL of sample off the BigDye XTerminator™ beads in the plate, then add 10 µL of 0.1 mM EDTA to dilute the sample. Re‑inject the sample using a standard run module (non‑BigDye XTerminator™ module). Decrease the injection voltage and injection time to match the BDX run module. Water was used as the injection solution. Repeat the sequencing reaction using less template. Use Hi‑Di™ Formamide or a 0.1 mM EDTA injection solution for samples. Note: Using water as an injection solution causes highly variable quantities of DNA to be injected, because there is no competition for the charged DNA/salts. 38 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

AAppendix A Troubleshooting Sanger sequencing data Poor 5′ sequencing quality Poor 5′ sequencing quality Although BigDye™ Terminator v3.1 and POP-7™ provide robust read lengths, properties of the dyes and polymer limit the ability to resolve the first 20– 25-bp in sequences. If a reading close to the first base following the sequencing priming region is required, an alternative approach would be to try BigDye™ Terminator v1.1 in combination with POP-6™ polymer or using the BigDye™ Direct Cycle Sequencing Kit. Example of poor 5′ sequencing quality Poor 5′ sequencing quality causes and actions Possible cause Recommended action This is normal within the first 35 bp when using POP-7™ polymer and BigDye™ Terminator v3.1 chemistry. To obtain sequence close to the primer, the use of POP-6™ polymer and the BigDye™ Terminator v1.1 Cycle BigDye XTerminator™ purification reagents exposed to Sequencing Kit, or the BigDye™ Direct Cycle Sequencing temperature over 25°C. Kit is recommended. Keep BigDye XTerminator™ purification reagents on ice if working with reagents for more than 30 minutes. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 39

B Select sequencing primers Primer considerations The following factors can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions with this kit: • Choice of sequencing primer sequence • Approach to primer purification These decisions are particularly important when sequencing is done on capillary electrophoresis systems where signal strength is critical. Some of the guidelines provided below are based on information that is general knowledge, while others are based on practical experience gained by our scientists. Primer guidelines The method of primer purification and choice of M13 tailed- or non-tailed sequencing primers can have a significant effect on the ease of reaction set-up and the quality of the sequencing data obtained in dye terminator cycle sequencing reactions. • HPLC-purification of all primers is recommended to minimize cycle sequencing noise and provide longer sequencing reads. • M13 sequencing primers are highly recommended because they facilitate the sequencing workflow set-up when sequencing multiple PCR products and they reduce the loss of valuable 5¢ unresolvable bases. With the M13 sequencing primers, you make single forward and reverse reaction mixes instead of pipetting a sequencing master mix followed by transfer of the appropriate forward/reverse PCR primers to each individual sequencing reaction. Note: The M13 forward or reverse sequence must be incorporated at the 5′ end of the PCR primer in order to use the M13 sequencing primers. Optimize primer selection Follow these guidelines to optimize primer selection: • Primers should be at least 18 bases long to: – Ensure good hybridization – Minimize the chance of having a secondary hybridization site on the target DNA 40 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

BAppendix B Select sequencing primers Primer Designer™ Tool • Avoid primers that have secondary structure or that can hybridize to form dimers. Several computer programs for primer selection are available. These programs help identify potential secondary structure problems and determine if a secondary hybridization site exists on the target DNA. • Avoid runs of an identical nucleotide, especially guanine, where runs of four or more Gs should be avoided. • Keep the G–C content in the 30–80% range. • For cycle sequencing, primers with melting temperatures (Tm) above 45°C produce better results than primers with lower Tms. • For primers with a G–C content < 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm > 45°C. Primer Designer™ Tool Primer Designer™ Tool is a free online tool to search for the appropriate PCR/Sanger primer pair from a database of >650,000 pre-designed primer pairs for resequencing the human exome. Go to: http://www.thermofisher.com/primerdesigner for more information, including a direct link to purchase the designed primers online. Primers ordered through the tool are free of known SNPs and primer-dimers, highly target-specific, and used under universal PCR conditions. Primers can be ordered unmodified, M13-tailed, HPLC-purified or desalted. The primers are checked by mass spectrometry and strict bioinformatics metrics, with bench validation showing a >95% success rate. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 41

C Partial sequence of pGEM™-3Zf(+) TGTAAAACGACGGCCAGT (–21 M13 forward primer) GAATTGTAAT ACGACTCACT ATAGGGCGAA TTCGAGCTCG 40 AGGCATGCAA 80 GTACCCGGGG ATCCTCTAGA GTCGACCTGC GCTTGGCGTA 120 TTGTTATCCG 160 GCTTGAGTAT TCTATAGTGT CACCTAAATA AGCATAAAGT 200 AACTCACATT 240 ATCATGGTCA TAGCTGTTTC CTGTGTGAAA GTCGGGAAAC 280 CAACGCGCGG 320 CTCACAATTC CACACAACAT ACGAGCCGGA CCGCTTCCTC 360 GCTGCGGCGA 400 GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT CGGTTATCCA 440 TGTGAGCAAA 480 AATTGCGTTG CGCTCACTGC CCGCTTTCCA GGCCGCGTTG 520 GACGAGCATC 560 CTGTCGTGCC AGCTGCATTA ATGAATCGGC GAAACCCGAC 600 TGGAAGCTCC 640 GGAGAGGCGG TTTGCGTATT GGGCGCTCTT CTTACCGGAT 680 TGGCGCTTTC 720 GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GGTGTAGGTC 760 CCCCCCGTTC 800 GCGGTATCAG CTCACTCAAA GGCGGTAATA ATCGTCTTGA 840 ACTGGCAGCA 880 CAGAATCAGG GGATAACGCA GGAAAGAACA TATGTAGGCG 920 ACTACGGCTA 960 AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA TCTGCTGAAG 1000 CTGGCGTTTT TCCATAGGCT CCGCCCCCCT ACAAAAATCG ACGCTCAAGT CAGAGGTGGC AGGACTATAA AGATACCAGG CGTTTCCCCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA AGCCCGACCG CTGCGCCTTA TCCGGTAACT GTCCAACCCG GTAAGACACG ACTTATCGCC GCCACTGGTA ACAGGATTAG CAGAGCGAGG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC 42 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

D Safety WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document. · Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device. · Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the “Documentation and Support” section in this document. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 43

D Appendix D Safety Chemical safety Chemical safety WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions: · Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the “Documentation and Support” section in this document. · Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). · Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). · Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer's cleanup procedures as recommended in the SDS. · Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) · After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. · Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. · IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. 44 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

DAppendix D Safety Biological hazard safety Biological hazard safety WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Safety equipment also may include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment. · U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at: www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf · World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at: www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 45

E Documentation and support Related documentation Document Publication number Description 4305080 DNA Sequencing by Capillary This chemistry guide is designed to Electrophoresis Chemistry Guide familiarize you with Applied Biosystems™ genetic analyzers for Generating high‑quality data using MAN0014628 automated DNA sequencing by the BigDye™ Terminator v3.1 Cycle capillary electrophoresis, to provide Sequencing Kit useful tips for ensuring that you obtain high‑quality data, and to help Troubleshooting Sanger sequencing MAN0014435 troubleshoot common problems. data This user bulletin provides guidance BigDye XTerminator™ Purification Kit 4374408 for generating high‑quality data using Protocol the BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide for 310 Genetic Analyzer Manual for 4317588 analysis with Minor Variant Finder Windows™ Software. Applied Biosystems™ 4477796 This document provides guidance for 3130/3130xl Genetic Analyzers the review of your data and Getting Started Guide troubleshooting tips for improving sequencing data quality. Describes protocols for BigDye XTerminator™purification This manual provides instructions for the set-up, operation, and maintenance of the 310 Genetic Analyzer. It also provides the instructions for setting up the Data Collection software for a run and contains additional information for instrument troubleshooting. This manual provides instructions for the set-up and operation of the 3130/3130xl Genetic Analyzer. It provides the instructions for setting up the Data Collection software, autoanalysis, monitoring and viewing data for a run. 46 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

EAppendix E Documentation and support Customer and technical support Document Publication number Description 3500/3500xL Genetic Analyzer with 100031809 This manual provides instructions for 3500 Series Data Collection Software the set-up, operation, maintenance 3.1 User Guide and troubleshootingof the 3500/3500xL Genetic Analyzer. It also User Guide: Applied Biosystems™ 4331468 provides the instructions for setting 3730/3730xl DNA Analyzer up the Data Collection software, analysis, monitoring and viewing data for a run. This manual provides instructions for the set-up and operation of the 3730xl DNA Analyzer. It provides the instructions for setting up the Data Collection software, autoanalysis, monitoring and viewing data for a run Portable document format (PDF) versions of this guide and the documents listed above are available at thermofisher.com. Customer and technical support Visit thermofisher.com/support for the latest in services and support, including: • Worldwide contact telephone numbers • Product support, including: – Product FAQs – Software, patches, and updates • Order and web support • Product documentation, including: – User guides, manuals, and protocols – Certificates of Analysis – Safety Data Sheets (SDSs; also known as MSDSs) Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer. Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale found on Life Technologies' website at www.thermofisher.com/us/en/home/global/ terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 47

Index 310 instruments quality 11 dye filter set 22 signal intensity 12 matrix standards 22 template quantity 12 documentation, related 46 3130 instruments dye sets, 3130, 3500, and 3730 instruments 22 dye set 22 dye/filter sets, 310 instrument 22 spectral standards 22 E 3500 instruments dye set 22 electrophoresis. See sample electrophoresis spectral standards 22 F 3730 instruments dye set 22 freeze-thaw cycles 7 spectral standards 22 I B instruments BAC DNA template, preparing 10 sequencing 8 bacterial artificial chromosomes. See BAC DNA tem- thermal cyclers 8 plate L biohazard safety 45 limited product warranty 47 C M calibration guidelines 22 capillary electrophoresis matrix standards, 310 instrument 22 310 Genetic Analyzer 23 P 3130/3130xl Genetic Analyzer 23 3500 /3500xL Genetic Analyzer 24 PCR template important reminders 21 importance of purifying 11 control DNA preparing 11 double-stranded 10 sequence 10, 42 pGEM-3Zf(+), partial sequence 42 single-stranded 10 prepare reactions for cycle sequencing 13 controls, use of 27 prepare templates cycle sequencing double-stranded 15 BAC 10 performing 13 double-stranded 10 prepare reactions for 13 PCR 11 run conditions 15 single-stranded 10 single-stranded 15 Primer Designer Tool 41 using buffer 14 primers, guidelines. See sequencing primers purify the sequencing reactions D BigDye XTerminator 16 DNA 48 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide

Centri-Sep plates 17 Index choose a method 16 ethanol/EDTA 19 signal intensity, and DNA template quantity 12 spectral standards, 3130, 3500 and 3730 instruments Q 22 quality of DNA 11 storing sequencing kits 7 R T reactions, prepare for cycle sequencing 13 template Ready Reaction format 6 determine quality 12 recommended raw signal ranges 27 determine quantity 12 related documentation 46 quality 11 required materials template preparation methods 10 electrophoresis 22 See also prepare templates sequencing 8 template quality 11 S templates, preparing 10 terms and conditions 47 safety, biohazard 45 troubleshooting sample electrophoresis 21 sequencing. See cycle sequencing dye blobs 35 sequencing buffer low signal 30, 31 mixed sequence throughout 32 optimized 6 mixed sequence up to or after a certain point 33 using 14 no signal 28 sequencing kits poor 5' sequencing quality 39 contents 7 poor mobility correction 34 storage conditions 7 signal saturation 37, 38 usage guidelines 7 sequencing primers U considerations 40 optimizing 40 usage guidelines, sequencing kits 7 selecting 40 W warranty 47 workflow 7 BigDye™ Terminator v3.1 Cycle Sequencing Kit User Guide 49

For support visit thermofisher.com/support or email techsupport@lifetech.com thermofisher.com 14 April 2016