Reagent Preparation Guide (for the Genome Analyzer)

TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) FOR RESEARCH USE ONLY Introduction 3 TruSeq SBS Kit v5 4 Preparing SBS Reagents 5 Reagent Kits and Replenishing Cycles 8 Technical Assistance ILLUMINA PROPRIETARY Catalog # FC-940-6001 Part # 15013595 Rev D November 2011

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Introduction This guide explains how to prepare reagents provided in the TruSeq SBS Kit v5 for sequencing runs on the Genome Analyzer. It contains an overview of kit contents and reagent preparation instructions. The hands-on time required for reagent preparation is about ten minutes for 36 cycles. However, runs longer than 36 cycles require that you prepare and combine reagents from multiple kits prior to each sequencing read. For information about setting up and performing a sequencing run, see one of the following guides depending on the version of Sequencing Control Software (SCS) you are running: } Genome Analyzer IIx User Guide (SCS v2.10), Part # 15030966 } Genome Analyzer IIx User Guide (SCS v2.9), Part # 15018814 Introduction Dual-ing on the Genome Analyzer This guide includes reagent information for dual-indexed sequencing runs, such as the required number of TruSeq SBS Kits and cycles when reagents must be replenished during a run. Dual-indexed sequencing is supported for sequencing of Illumina Nextera libraries only. You must run Sequencing Control Software (SCS) v2.10 and v10 dual-indexing recipes. Sequencing of Illumina Nextera libraries requires specific sequencing primers provided in the TruSeq Dual Sequencing Primers Box, in addition to the indexing and paired-end reagents provided in the TruSeq Cluster Kit. These primers are required for Read 1 (applied during cluster generation), the Reads (single or dual), and Read 2. For more information, see the Genome Analyzer IIx User Guide (SCS v2.10), Part # 15030966. Reagent Storage The TruSeq SBS Kit v5 is shipped on dry ice in a single package containing two boxes. When you receive your kit, store the kit components according to the temperature listed on the box label. Components from one box are stored at 2 to 8 C, and components from the other box are stored at -15 to -20 C. TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 3

TruSeq SBS Kit v5 All reagents provided in the TruSeq SBS Kit are labeled with the prefix GA plus a barcode number that uniquely identifies the container. SBS Box 1: Store at 2 to 8 C Reagent Name PW1 PR1 PR2 PR3 Reagent Description Wash Solution High Salt Buffer Incorporation Buffer Cleavage Buffer SBS Box 2: Store at -15 to -25 C Reagent Name IMR LFN HDP CLM SMX Reagent Description Incorporation Mix Reagent Long-Read FFN High Density Polymerase Cleavage Mix Scan Mix The SMX instrument tube is not required for use on the GAIIx. 4 Part # 15013595 Rev D

Preparing SBS Reagents One kit provides reagents for 36 cycles of sequencing. For runs longer than 36 cycles, you must prepare reagents from multiple kits. For more information, see Reagent Kits and Replenishing Cycles on page 8. Reagent Containers Bottle Positions and Reagents Catalog # 175 ml Bottle Positions 1, 3, and 6 (IMR, SMX, and CLM) 175 ml bottles hold sufficient reagents for up to 151 cycles of sequencing. BD Catalog # 352076 Preparing SBS Reagents 125 ml Bottle Positions 4, 5, and 7 (PR1, PR2, and PR3) 125 ml Nalgene bottles connect to threaded bottle receptacles on the instrument. SBS reagents are kitted in 125 ml bottles. ThermoFisher Scientific Catalog # 342040-0125 150 ml Bottles Positions 4, 5, and 7 (PR1, PR2, and PR3) [Optional/User-Supplied] 150 ml bottles minimize the need to replenish PR1, PR2, and PR3 during the run. Remove the threaded bottle adapter before loading 150 ml bottles. Corning Catalog # 431175 TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 5

Thaw Reagents For paired-end runs, thaw reagents for Read 1 and any indexing cycles first, and thaw reagents for Read 2 only when you are ready to prepare them fresh just prior to the start of Read 2. 1 Remove IMR, LFN, and SMX from -15 to -25 C storage, and thaw at room temperature or in a beaker containing deionized water for about one hour. Leave the HDP in -15 to -25 C storage until you are ready to use it to prepare the IMR. CAUTION It is important to keep the CLM away from the other components to avoid cross-contamination. After handling the CLM container, be sure to discard your gloves and replace them with a new pair each time. 2 Remove the CLM from -15 to -25 C storage and thaw it at room temperature or in a separate beaker containing deionized water for about one hour. 3 Record the lot numbers of each reagent on the lab tracking form. 4 Immediately after the reagents have thawed, place them on ice. Be sure to keep the CLM in a separate ice bucket during reagent preparation. Prepare PR1, PR2, and PR3 1 Invert the bottle of PR1 several times to mix the reagent. 2 Invert the bottle of PR2 several times to mix the reagent. 3 Invert the bottle of PR3 several times to mix the reagent. 4 When preparing multiple kits, combine reagents in either a 125 ml bottle or an optional user-supplied 150 ml bottle. 5 Set aside excess PR1, PR2, and PR3 at room temperature to replenish during the run. Prepare IMR 1 Pour the contents of the IMR container into one 175 ml Falcon bottle. 6 Part # 15013595 Rev D

2 Transfer 3.52 ml of LFN to the IMR reagent. 3 Remove the HDP tube from -15 to -25 C storage and briefly pulse centrifuge. 4 Transfer 330 µl of HDP into the IMR/LFN mix. 5 Cap the bottle of IMR mix tightly and invert five times to mix. 6 Centrifuge at 1,000 xg for one minute at 4 C. 7 When preparing multiple kits, combine up to 185 ml of IMR into one 175 ml bottle. 8 Place the IMR on ice until you are ready to load reagents. Prepare SMX 1 Invert the bottle of SMX several times to mix well. 2 Pour the SMX into one 175 ml Falcon bottle. 3 If your run requires more than three kits, use SMX from a fourth kit to fill the bottle with 185 ml of SMX. 4 Cap the container and place the SMX on ice until you are ready to load reagents. 5 Set aside excess SMX at 2 to 8 C to replenish reagent during the run. Preparing SBS Reagents Prepare CLM 1 Invert the CLM tube several times to mix well. 2 Centrifuge at 1,000 xg for one minute at 4 C. 3 Pour the contents of the CLM container into one 175 ml Falcon bottle. 4 If your run requires more than four kits, use CLM from a fifth kit to fill the bottle with 185 ml of CLM. 5 Cap the container and place the CLM in a separate ice bucket until you are ready to load reagents. 6 Discard your gloves and replace them with a new pair. TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 7

Reagent Kits and Replenishing Cycles This section provides the number of TruSeq SBS Kits required for each reagent used in a sequencing run of various lengths and run types, and the required cycles to replenish reagents. If you are using a Genome Analyzer II, the preparation of reagents and schedule for replenishing reagents differs for positions 1, 3, and 6. For more information, see the Genome Analyzer IIx User Guide. Using the Reagent Kits and Replenishing Cycles Tables } The number of required kits are listed for the first read and any indexing cycles. The number of required kits for Read 2 are listed separately. Always prepare fresh SBS reagents for Read 2. } The "X" indicates which reagents you need to replenish at the indicated cycles. } The "X" at the start of Read 1 and start of Read 2 indicates that a full set of fresh reagents are required before starting the read. } For single-read runs, disregard the Read 2 portion of the table. } For dual-indexed runs, the seven chemistry-only cycles performed during the 2 (i5) Read are calculated for reagent use; however, these cycles are not counted in the total number of cycles performed in the run. 151-Cycle Non-ed Run Cycle 105 108 X X X Cycle 151 Cycle 152 Cycle 256 259 X X X Cycle 302 8 Part # 15013595 Rev D

} SMX Combine 185 ml of prepared SMX. Discard the remaining reagent. reagent. Set aside remaining reagent to replenish during the run. Discard the remaining reagent. 126-Cycle Non-ed Run Cycle 105 108 X X X Cycle 126 Cycle 127 Cycle 231 234 X X X Cycle 252 } SMX Combine 185 ml of prepared SMX. Discard the remaining reagent. reagent. Set aside remaining reagent to replenish during the run. Discard the remaining reagent. Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 9

101-Cycle Non-ed Run Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 101 Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 102 Cycle 202 reagent. Discard the remaining reagent. Discard the remaining reagent. 51-Cycle Non-ed Run Number of SBS Kits: 2 2 2 2 1 2 2 1 2 51 Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 52 102 reagent. Discard the remaining reagent. 10 Part # 15013595 Rev D

36-Cycle Non-ed Run Number of SBS Kits: 1 1 1 1 1 1 1 1 1 Cycle 36 Number of SBS Kits: 1 1 1 1 1 1 1 1 1 Cycle 37 Cycle 72 151-Cycle Single-ed Run Number of SBS Kits: 4 4 5 3 2 3 3 2 3 Cycle 105 108 X X X Cycle 151 X X X X Read Cycle 152 Cycle 159/160 Cycle 264 267 X X X Cycle 309/310 Reagent Kits and Replenishing Cycles } SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. } CLM Combine 185 ml of prepared CLM. Discard remaining reagent. TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 11

reagent. Set aside remaining reagent to replenish during the run. Set aside remaining reagent to replenish. 146-Cycle Single-ed Run Cycle 105 108 X X X Cycle 146 X Read Cycle 147 Cycle 154/155 Cycle 259 262 X X X Cycle 299/300 } SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. reagent. Set aside remaining reagent to replenish during the run. Discard remaining reagent. 12 Part # 15013595 Rev D

126-Cycle Single-ed Run Cycle 105 108 X X X Cycle 126 Read Cycle 127 Cycle 134/135 Cycle 238 241 X X X Cycle 259/260 } SMX Combine 185 ml of prepared SMX. Discard remaining reagent. reagent. Set aside remaining reagent to replenish during the run. Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 13

101-Cycle Single-ed Run Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 101 Read Cycle 102 Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 109/110 Cycle 209/210 reagent. Discard remaining reagent. 51-Cycle Single-ed Run Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 51 Read Cycle 52 Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 59/60 Cycle 109/110 14 Part # 15013595 Rev D

reagent. Discard remaining reagent. 36-Cycle Single-ed Run Number of SBS Kits: 1 1 1 1 1 1 1 1 1 Cycle 36 Read Cycle 37 Number of SBS Kits: 1 1 1 1 1 1 1 1 1 Cycle 43/44 Cycle 79/80 151-Cycle Dual-ed Run Number of SBS Kits: 4 5 5 3 2 3 3 2 3 Cycle 105 108 X X X Cycle 151 X X X X Reads Cycle 152 Cycle 168 Cycle 272 275 X X X Cycle 318 Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 15

} SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. } CLM Combine 185 ml of prepared CLM. Discard remaining reagent. reagent. Set aside remaining reagent to replenish during the run. Set aside remaining reagent to replenish. 146-Cycle Dual-ed Run Number of SBS Kits: 4 5 5 3 2 3 3 2 3 Cycle 105 108 X X X Cycle 146 X X X X Reads Cycle 147 Cycle 163 Cycle 267 272 X X X Cycle 308 } SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. } CLM Combine 185 ml of prepared CLM. Discard remaining reagent. reagent. Set aside remaining reagent to replenish during the run. Set aside remaining reagent to replenish. 16 Part # 15013595 Rev D

133-Cycle Dual-ed Run Cycle 105 108 X X X Cycle 133 X Reads Cycle 134 Cycle 150 Cycle 254 257 X X X Cycle 282 } SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. reagent. Set aside remaining reagent to replenish during the run. Discard remaining reagent. Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 17

126-Cycle Dual-ed Run Cycle 105 108 X X X Cycle 126 X Reads Cycle 127 Cycle 143 Cycle 238 241 X X X Cycle 268 } SMX Combine 185 ml of prepared SMX. Set aside remaining reagent to replenish. reagent. Set aside remaining reagent to replenish during the run. Discard remaining reagent. 18 Part # 15013595 Rev D

101-Cycle Dual-ed Run Number of SBS Kits: 3 4 4 3 2 3 3 2 3 Cycle 101 Reads Cycle 102 Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 118 Cycle 218 } SMX Combine 185 ml of prepared SMX. Discard remaining reagent. reagent. Discard remaining reagent. Discard remaining reagent. Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 19

93-Cycle Dual-ed Run Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 93 Reads Cycle 94 Number of SBS Kits: 3 3 3 3 2 3 3 2 3 Cycle 110 Cycle 202 reagent. Discard remaining reagent. Discard remaining reagent. 20 Part # 15013595 Rev D

51-Cycle Dual-ed Run Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 51 Reads Cycle 52 Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 68 Cycle 118 reagent. Discard remaining reagent. Discard remaining reagent. Reagent Kits and Replenishing Cycles TruSeq SBS Kit v5 Reagent Preparation Guide (for the Genome Analyzer) 21

36-Cycle Dual-ed Run Number of SBS Kits: 2 2 2 2 1 2 2 1 2 Cycle 36 Reads Cycle 37 Number of SBS Kits: 1 1 1 1 1 1 1 1 1 Cycle 52 Cycle 88 reagent. Discard remaining reagent. Discard remaining reagent. 22 Part # 15013595 Rev D

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