├── MiSeq_WetLab_SOP.md └── Schloss.txt /MiSeq_WetLab_SOP.md: -------------------------------------------------------------------------------- 1 | # MiSeq Wet Lab SOP 2 | 3 | 4 | **Detailed Title:** | 16S rRNA Sequencing with the Illumina MiSeq: Library Generation, QC, & Sequencing 5 | **Authors:** | James Kozich, Patrick Schloss, Niel Baxter, Matt Jenior, Charles Koumpouras, Lucas Bishop 6 | **Original Date:** | 25-March-2013 7 | **Version:** | 6.0 8 | **Last Updated:** | 8-August-2019 9 | **Updated By:** | Lucas Bishop 10 | 11 | 12 | ------------------------------------ 13 | **1.0) Introduction and Workflow** 14 | ------------------------------------ 15 | 16 | ***1.1) Introduction*** 17 | 18 | - The Purpose of this protocol is to define the steps for 19 | the preparation and sequencing of 16S rRNA gene sequence libraries 20 | using the Illumina MiSeq sequencing platform, as described in the paper 21 | [Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform](http://www.ncbi.nlm.nih.gov/pubmed/23793624) by Kozich et al. 22 | 23 | - The Illumina MiSeq Personal Sequencer can produce 2 x 250bp (or 2 x 300bp with v3 chemistry) 24 | paired-end reads and up to 8.5 Gb of data in a single run. Dual indexing of 25 | library samples allows up to 384 samples to be run simultaneously. 26 | The instrument is capable of producing in excess of 24 million 27 | reads. However for low diversity runs about 12 million reads can be 28 | expected. A wide range of applications is possible including 16S 29 | analysis, metagenomics, genome sequencing, transcriptomics, and RNA 30 | sequencing. 31 | 32 | - Our lab typically sequences the V4 region of the 16S rRNA gene. 33 | Its short length (~250bp) allows for fully overlapping forward and reverse reads, 34 | which, in combination with our [curaton pipeline](http://www.mothur.org/wiki/MiSeq_SOP), 35 | results in the lowest error rates. We also prefer to use the older v2 MiSeq cartridges, as the 36 | newer v3 chemistry consistently results in a higher error rates in our hands. 37 | 38 | - There are several steps in preparing samples for sequencing on the 39 | MiSeq. Broadly, these include library generation and indexing, 40 | quality control, normalization and pooling, quantification, 41 | sequencing, run quality assessment, and data export. An overview of 42 | each step and more detailed protocols are below. 43 | 44 | ***1.2) 16S Prep Workflow*** 45 | 46 | This section is an overview of the steps involved in library preparation. 47 | For a more detailed desctiption of the methods, see Section 5 below. 48 | 49 | 1. Extracted DNA should be arrayed in 96 well plate format, preferably with 50 | two wells on each plate open for controls. We use the Qiagen DNeasy PowerSoil HTP 96 Kit (Cat# 12955-4) for all extractions. 51 | 52 | 2. Samples are PCR amplified with Schloss lab indices. Each plate should contain a negative control 53 | (water) and a positive control (mock community) 54 | 55 | 3. A subset of 12-24 samples from each plate undergoes electrophoresis 56 | on a 1% agarose gel to ensure amplification proceeded normally. 57 | 58 | 4. Library clean up and normalization is performed using the Invitrogen 59 | [SequalPrep](http://products.invitrogen.com/ivgn/product/A1051001) 60 | Plate Normalization Kit. 61 | 62 | 5. Samples from each plate are pooled into single wells (i.e. 1 well/plate). 63 | 64 | 6. (Optional) To assess the quality of the library, the investigator may choose to perform 65 | a Bioanalyzer trace using the [Agilent Technologies HS DNAkit](http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1635) cat\# 5067-4626. 66 | 67 | 7. (Optional) If the post-PCR gel or the Bioanalyzer trace suggests contaminant DNA 68 | from leftover indices/primer-dimer, an additional gel purification of the pooled 69 | plates is recommended. This often improves the quality of the sequencing run. 70 | 71 | 8. Each pooled plate is quantified using a KAPA Biosystems [qPCR kit](http://www.kapabiosystems.com/products/name/kapa-library-quant-kits) cat\# KK4824. 72 | 73 | 9. Plates are pooled to equal concentration into a single well (i.e. 1 well per run) 74 | 75 | 10. The pooled library enters the Sequencing Workflow. 76 | 77 | ***1.3) Sequencing Workflow*** 78 | 79 | This section is an overview of the steps involved in initiating a sequencing run. 80 | For a more detailed desctiption of the mehods, see Section 5 below. 81 | 82 | 1. A Sample Plate is created for each plate using Illumina Experiment Manager. 83 | Sample Plates are then used to create a Sample Sheet. This sheet serves 84 | as the set of run parameters and indexing scheme used by the MiSeq for the run. 85 | The sample sheet is then transferred to the MiSeq via flash drive. 86 | 87 | 2. The reagent cartridge is thawed in a water bath per the [MiSeq System Guide](https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/miseq-system-guide-15027617-01.pdf). 88 | 89 | 3. Unless otherwise specified, dilution and loading will follow the 90 | steps outlined in the document: [Preparing DNA Libraries for Sequencing on the MiSeq](https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/preparing-libraries-for-sequencing-on-miseq-15039740-d.pdf) 91 | a. Pooled library and PhiX control are denatured and diluted. 92 | b. Diluted library and PhiX are pooled (10-15% PhiX, 85-90% Library). 93 | c. Sequencing primers and library/PhiX are loaded into the reagent cartridge. 94 | d. MiSeq flow cell is washed. 95 | 96 | 4. The sample sheet, flow cell, reagent cartridge, PR2 bottle, and an empty waste bottle 97 | are loaded onto the MiSeq, and the run is initiated. A 500 cycle run takes approx. 44 hours. 98 | 99 | 5. The run is monitored using Illumina Sequence Analysis Viewer. 100 | 101 | 6. Upon completions of the run, fastq files are transfered to the Schloss Lab NAS drive. 102 | 103 | 7. A post run wash is performed, followed by a standby wash if the machine will be idle for a week or more. 104 | a. Between sequencing runs we recommened a bleach wash which is part of the post run wash. 105 | - 0.5% Tween20 in reservoir bottle per usual. 106 | - 6 mL of 0.5% Tween20 to each cartridge well except 17. 107 | - Mix 870uL of ultrapure H2O AND 30uL of 6% Bleach to a 1.5 mL tube. 108 | - Note: This is to be made fresh for each wash. 109 | - Mix 50uL of the bleach/water solution above and 950uL of ultrapure H2O in the MiSeq wash tube. 110 | - Place the MiSeq wash tube in cartridge well 17. Load the cartridge into the MiSeq. 111 | - Start a post run wash on the MiSeq with “template line wash” selected. 112 | ------------------------------------ 113 | **2.0) Safety and Waste Disposal** 114 | ------------------------------------ 115 | 116 | - The Schloss Lab Chemical Hygiene Plan should be followed at all 117 | times. 118 | 119 | - Standard PPE (nitrile gloves, safety glasses, and lab coat) should 120 | be used at all times. 121 | 122 | - Each reagent cartridge contains a small amount of formamide and must 123 | be disposed of in an appropriate container following the run. Liquid 124 | waste from a run must also be disposed of as hazardous due to the 125 | formamide content. 126 | 127 | 128 | ---------------------- 129 | **3.0) Consumables** 130 | ---------------------- 131 | 132 | Reagent/Kit | Catalog \# | Price 133 | -------------------------------------------------------|-------------|----------- 134 | MiSeq® Reagent Kit v2 (500 cycle) | MS-102-2003 | $1016.40 135 | PhiX Control Kit v3 | FC-110-3001 | $134.40 136 | 16s Index Primers (40 total) | IDT |$1180.00 137 | 16s Sequencing Primers (Read 1, Index, Read 2) | IDT | $33.25 138 | AccuPrime™ Pfx SuperMix | 12344-040 | $462.60 139 | AccuPrime™ Pfx DNA polymerase (200 rxns) | 12344-024 | $402.00 140 | KAPA Lib. Quantification Kit - Illumina/Universal | KK4824 | $751.98 141 | SequalPrep™ Normalization Plate (96) Kit | A10510-01 | $1494.00 142 | (optional) Agilent High Sensitivity DNA Kit | 5067-4626 | $434.70 143 | epT.I.P.S. Motion 50 uL Filter Reloads 24 racks | 30014430 | $391.00 144 | epT.I.P.S. Motion 1000 uL Filter Reloads 24 racks | 30014510 | $391.00 145 | Tip One Refill Wafers 200uL elongated graduated | NC9549602 | $39.24 146 | Tip One Refill Wafers 1000uL graduated | 1111-2721 | $25.25 147 | Fisher 1N NaOH 1L | SS266-1 | $27.61 148 | TWIN.TEC 96 Well Plate Skirted Clear | E951020401 | $102.09 149 | MiSeq Disposable Wash Tubes | 15054695 | ~$5.00 150 | 151 | -------------------- 152 | **4.0) Run Costs** 153 | -------------------- 154 | 155 | For 384 sample run | PCR and Indexing | Cleanup & Normalization | Library QC | Sequencing | Totals 156 | ----------------- | --------------- | ----------------------| --------- | ---------- | ------ 157 | 16S Reagents | $610 | $598 | $138 | $1120 | $2466 158 | 16S Man hours | 4 | 3 | 4 | 4 | 15 159 | 160 | -------------------- 161 | **5.0) Detailed Method(s)** 162 | -------------------- 163 | 164 | ***5.1) Published Protocols*** 165 | 166 | - The following methods and references are used in the workflows 167 | above. 168 | 169 | - [MiSeq System Guide](https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/miseq-system-guide-15027617-01.pdf) 170 | 171 | - [Preparing DNA Libraries for Sequencing on the MiSeq](https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/preparing-libraries-for-sequencing-on-miseq-15039740-d.pdf) 172 | 173 | - Kapa Biosystems qPCR Library Quantification Kit Illumina 174 | 175 | - [Accuprime Pfx Super Mix](http://tools.invitrogen.com/content/sfs/manuals/accuprimepfxsupermix_man.pdf) 176 | 177 | - [SequalPrep Normalization Plate (96) Kit](http://tools.lifetechnologies.com/content/sfs/manuals/sequalprep_platekit_man.pdf) 178 | 179 | - [Agilent High Sensitivity DNA Kit Guide](http://www.chem.agilent.com/library/usermanuals/Public/G2938-90321_SensitivityDNA_KG_EN.pdf) 180 | 181 | - [QIAquick Gel Extraction Kit](https://www.qiagen.com/us/resources/download.aspx?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en) 182 | 183 | 184 | ***5.2) Initial Set up*** 185 | 186 | 1. Reconstitute indexed primers and sequencing primers to 100 uM. See 187 | Appendix D for primer design. 188 | 189 | 2. Prepare 100ul 10 uM aliquots of indexed primers. Do not dilute 190 | sequencing primers. 191 | 192 | 3. Array equal aliquots of indexed primers into four 96 well plates using the following scheme: 193 | 194 | a. A701 – A712 with A501 – A508 195 | b. A701 – A712 with B501 – B508 196 | c. B701 – B712 with B501 – B508 197 | d. B701 – B712 with A501 – A508 198 | 199 | Note: These primer plates can be stored at -20°C and used for subsequent runs. 200 | 201 | 4. Extract template DNA and array in 96 well format leaving two wells 202 | open. (One for a negative water control and another for the positive 203 | Mock Community control) 204 | 205 | 4b. (OPTIONAL) Quantify the extracted DNA to confirm acceptable amounts are present for the PCR. 206 | 207 | 5. Using Illumina Experiment Manager software (only compatible with windows), create a sample plate for each 96 208 | well plate of template. Choose indexes that correspond to one of the 209 | four index pair plates above. See Appendix A for instruction on 210 | creating a custom assay in IEM. 211 | 212 | 6. Using Illumina Experiment Manager, create a sample sheet for the 213 | run. Ensure that index choices are compatible with one another and 214 | there is sufficient diversity in the index reads so as to activate 215 | both light channels every cycle. Note: Primer plate A has 216 | insufficient diversity to be run alone. If sequencing 96 or fewer 217 | samples, choose plate B, C, or D. 218 | 219 | 220 | ***5.3) PCR*** 221 | 222 | Note: These steps may be performed using an epMotion or similar 223 | automated pipetting system. 224 | 225 | 1. Dispense 17 ul of Accuprime Pfx Supermix into each well of a new 96 226 | well plate. 227 | 228 | 2. Using a multichannel pipette, transfer 1 ul of template DNA per well 229 | to the corresponding well on the PCR plate. 230 | 231 | 3. Using a multichannel pipette, transfer 2 ul of each paired set of 232 | index primers from the primer plate to the corresponding well on the PCR plate. Be sure to 233 | follow the layout chosen in the sample sheet. 234 | 235 | 4. Add 1 ul of PCR grade H~2~O to the negative control well, and 1 ul 236 | of Mock Community at a 1:3 dilution to the positive control well. 237 | 238 | 5. Repeat for up to four 96 well plates. Seal plates, vortex briefly 239 | and spin down contents. 240 | 241 | 6. Place in thermocycler. 242 | 243 | Use the following program: 244 | 245 | > 95°C 2:00 246 | > --------30 cycles-------- 247 | > 95°C 00:20 248 | > 55°C 00:15 249 | > 72°C 5:00 250 | > \---------------------- 251 | > 72°C 10:00 252 | > 4°C Hold 253 | 254 | 255 | ***5.4) Gel Electrophoresis*** 256 | 257 | 1. 1 or 2 random rows of 12 should be selected from each PCR plate and run on 258 | a gel to confirm success of the PCR. (Alternatively all samples can be run on a single invitrogen E-Gel) 259 | 260 | 2. Use 4 ul of sample, 4 ul of loading dye in a 1% agarose gel. 261 | 262 | 3. Run at 100v for 60 minutes alongside a 1kb+ ladder or E-Gel low range ladder. 263 | 264 | 4. Photograph gel under UV. Check to be sure there is a band for every 265 | well. 266 | 267 | 268 | ***5.4b) Troubleshooting*** 269 | - Sometimes not every well from the PCR plate will result in a band. 270 | 271 | 1. Locate wells in the PCR plate that did not result in a band on the gel and mark which extracted DNA samples they correspond to. 272 | 273 | 2. Using a pipette, aspirate and discard the PCR product from the wells that did not produce bands. 274 | 275 | 3. Set up a new 20 ul PCR reaction, this time using : 276 | - 7 ul of template DNA 277 | - 2 ul of the SAME paired primers that were used originally for that sample 278 | - 11 ul of mastermix made with the non-supermix polymerase (Cat.# 12344-24). Reference the product sheet when making the mastermix to account for change in the amount of input DNA. 279 | 280 | 4. Place in thermocycler and use the same cycling conditions as stated in section 5.3. 281 | 282 | 5. Re-run on a gel alongside a 1kb+ ladder or E-Gel low range ladder. 283 | - For E-gels: Every well needs liquid in it (use water blanks in empty wells) for gel to work. 284 | 285 | 6. Photograph gel under UV to check that there are bands for every PCR reaction. 286 | 287 | 7. Using a pipette, transfer the re-done PCR product to the original PCR plates. 288 | 289 | 290 | ***5.5) Cleanup, Normalization, and Pooling*** 291 | 292 | Use the SequalPrep Normalization Plate Kit. These steps can be automated on an epMotion system. 293 | 294 | 1. Transfer 15 ul of PCR product from PCR plate to corresponding well 295 | on the normalization plate. 296 | 297 | 2. Add 15 ul of Binding Buffer. Mix by pipetting, sealing, vortexing, 298 | and spinning briefly. 299 | 300 | 3. Incubate at room temperature for 60 minutes. Plate can be incubated 301 | overnight at 4C if needed. Extra time does not improve results. 302 | 303 | 4. Aspirate the liquid from the wells. Do not scrape the sides. 304 | 305 | 5. Add 50 ul of Wash Buffer and pipette up and down twice, then 306 | aspirate immediately. Invert and tap plate on a paper towel to 307 | Ensure there is no residual wash buffer in any wells. 308 | 309 | 6. Add 20 ul of Elution Buffer. Mix by pipetting up and down 5 times. 310 | Seal, vortex, and spin briefly. 311 | 312 | 7. Incubate at room temperature for 5 minutes. 313 | 314 | 8. Create a pool from each plate. Take 5 ul of each well to pool. The 315 | use of an empty 96 well plate may facilitate the use of multichannel 316 | pipettes. 317 | 318 | 9. Freeze the remaining sample for later use. 319 | 320 | ***5.6) Library QC & Quantification*** 321 | 322 | 1. Prepare the following dilutions of each pooled library in PCR grade 323 | H~2~O (or 10nM Tris-HCl + 0.05% Tween20): 324 | 325 | a. 1:1 326 | 327 | b. 1:10 328 | 329 | c. 1:1000 (dilute in several steps for better results) 330 | 331 | d. 1:2000 332 | 333 | e. 1:4000 334 | 335 | f. 1:8000 336 | 337 | 2. (Optional) Agilent Bioanalyzer Trace 338 | 339 | a. Prepare Gel-Dye mix if not already prepared. 340 | 341 | b. Let reagents equilibrate to room temperature. 342 | 343 | c. Turn Bioanalyzer on and load 350 ul of dH~2~O onto electrode 344 | cleanser and place in analyzer for 5 minutes. 345 | 346 | d. Open a high sensitivity chip and place on the priming station. 347 | Base plate should be a position “C” and syringe clip should be 348 | at lowest position. 349 | 350 | e. Load 9.0 ul of gel-dye mix to position 12 market with a large 351 | “G”. Ensure the syringe plunger is at 1.0 ml and close the 352 | station. Press plunger until it is held by clip. 353 | 354 | f. Wait for exactly 60 seconds then release the plunger clip. Wait 355 | an additional 5 seconds, then slowly pull the plunger back to 356 | the 1.0 ml position. 357 | 358 | g. Open the priming station. Pipette 9.0 ul of gel-dye mix into the 359 | other wells marked “G” in positions 4,8,and 16. 360 | 361 | h. Pipette 5.0 ul of marker to all wells excluding the right 362 | column. (No marker positions 4,8,12, and 16) 363 | 364 | i. Load 1 ul of ladder into position 15 marked by the ladder 365 | symbol. 366 | 367 | j. Pipette 1 ul of each of dilutions a – b above. Top row Plate 1 368 | Pool 1:1 x 1, 1:10 x 2. Second row Plate 2 pool 1:1 x 1, 1:10 x 369 | 2. Third row Plate 3 Pool 1:1 x 1, 1:10 x 2. Bottom row Plate 4 370 | Pool 1:1 x 1, 1:10 x 2. 371 | 372 | k. Place chip in the designated vortex for 1 minute, then transfer 373 | chip to the Bioanalyzer. 374 | 375 | l. Open the 2100 Expert software and select the HS DNA Assay. Enter 376 | sample names/dilutions for each of the test wells. Click Start. 377 | 378 | m. Print .pdf when run finishes. 379 | 380 | 3. (Optional) Gel Purification 381 | 382 | a. Load 50-200ul of each pooled plate on a 1% agarose gel. It will usually be necessary to 383 | tape several combs together to accomadate the volume. 384 | 385 | b. Run the gel for ~1.5hr at 80V, until there is sufficient separation 386 | between the amplicon and the indices. 387 | 388 | c. Briefly image the gel under UV light to locate and excise the band. 389 | 390 | b. Follow the manufactures protocol for extracting the DNA from the gel ([QIAquick Gel Extraction Kit](https://www.qiagen.com/us/resources/download.aspx?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en)). 391 | 392 | c. Once the DNA is isolated, proceed to qPCR quantification. 393 | 394 | 395 | 4. Kapa qPCR Library Quantification 396 | 397 | a. Before qPCR reaction setup, add 1 ml Primer Premix (10X) to the 398 | 5 ml bottle of KAPA SYBR® FAST qPCR Master Mix (2X) and mix by 399 | vortexing for 10 sec. Record the date of Primer Premix addition 400 | on the KAPA SYBR® FAST qPCR Master Mix bottle. 401 | 402 | b. Reaction can be either 10 ul or 20 ul. A 10 ul reaction volume 403 | is recommended. 404 | 405 | c. Prepare a 96 well qPCR plate compatible with the real time 406 | thermocycler. There are six standards. Each should be run in 407 | triplicate. Each pool at each dilution should be run in 408 | triplicate. 409 | 410 | d. For 10 ul reaction volume dispense 6 ul of master mix into each 411 | well needed. 412 | 413 | e. Pipette 4 ul of standards and library dilutions into appropriate 414 | wells. Mix by pipetting. Vortex and spin optional. 415 | 416 | f. Place plate in thermocycler. Start control software 417 | 418 | g. Program the following cycle 419 | 420 | i. Initial Activation 95°C 5 minutes 421 | 422 | ii. 35 cycles 423 | 424 | 1. Denaturation 95°C 30 seconds 425 | 426 | 2. Annealing 60°C 45 seconds 427 | 428 | 3. If library fragment size exceeds 700bp, extend annealing 429 | step to 90 seconds. 430 | 431 | iii. Perform melt curve to check for primer/adaptor dimer 432 | 433 | h. Assign wells and group replicates. 434 | 435 | i. Enter values for standards 436 | 437 | i. Std. 1 20pM 438 | 439 | ii. Std. 2 2pM 440 | 441 | iii. Std. 3 0.2pM 442 | 443 | iv. Std. 4 0.02pM 444 | 445 | v. Std. 5 0.002pM 446 | 447 | vi. Std. 6 0.0002pM 448 | 449 | vii. Note: The concentrations provided here are for the DNA 450 | Standards as supplied in the kit, and are NOT the 451 | concentrations in the reactions. Provided that the volume of 452 | template added to each reaction is the same for Standards 453 | and for library samples (i.e. 4 ul in each case), there is 454 | no need to account for these volumes when calculating the 455 | concentrations of library samples, nor should one need to 456 | calculate the concentration of template in the reaction. 457 | 458 | j. Run program 459 | 460 | k. To calculate library concentration use the following formula: 461 | 462 | i. Average x (452/Avg fragment length from bioanalyzer) x 463 | dilution factor 464 | 465 | ii. Use the average of the triplicate data points corresponding 466 | to the most concentrated library DNA dilution that falls 467 | within the dynamic range of the DNA Standards to calculate 468 | the concentration of the undiluted library. 469 | 470 | iii. Do not include outliers in calculation. If there is more 471 | than one outlier in a group, the assay must be repeated. 472 | 473 | 5. Create a single final library by pooling each of the 4 pooled plates into a single 474 | well. Be sure to pool such that each plate has an equal final conentration (not necessarily equal volumes. 475 | Hint: C~1~ V~1~ = C~2~ V~2~ ). Final pool must be \>10ul in total volume (40-80ul of \>1nM library is ideal) 476 | 477 | ***5.7) Sequencing*** 478 | 479 | 1. Remove a 500 cycle reagent cartridge from the -20°C freezer. 480 | Place in room temperature water bath for one hour. Place Hyb buffer 481 | tube in 4°C fridge. While reagent cartridge is thawing, perform steps 2-6. 482 | 483 | 2. Prepare fresh 0.2N NaOH. 484 | 485 | 3. To a 1.5ml tube, add 10 ul of library and 10 ul of 0.2N NaOH. To a 486 | separate tube add 2 ul PhiX, 3 ul PCR grade water, and 5 ul of 0.2N 487 | NaOH. Pipette to mix. Note: NaOH concentration on the flow cell must 488 | remain under 0.001N. Adjusting the concentration of the NaOH used to 489 | denature the DNA to 0.1N may be necessary if library concentration is 1nM or below.[^1] 490 | 491 | 4. Allow the tubes to incubate at room temperature for 5 minutes. 492 | Immediately add 980 ul of ice-cold Hyb to the library tube, 493 | and 990 ul Hyb to the PhiX tube. Note: the resulting 20pM PhiX can be 494 | frozen and used for subequent runs. 495 | 496 | 5. Use Hyb to further dilute both the library and PhiX to 4pM for a v2 kit. Can 497 | load up to 8pM for a v3 kit. 498 | 499 | See example below: 500 | a. (1.45 nM library x 10 ul) + (0.2N NaOH x 10 ul) + 980 ul Hyb = 501 | 14.5pM Lib, 0.002N NaOH 502 | 503 | b. (14.5pM lib x 275.86 ul) + 724.14 ul Hyb = 4.0pM lib, 0.00055N 504 | NaOH 505 | 506 | c. [(10nM PhiX x 2 ul) + 3 ul H~2~O] + (0.2N NaOH x 5 ul) + 990 ul 507 | Hyb = 20pM PhiX, 0.001N NaOH 508 | 509 | d. (20pM PhiX x 200 ul) + 800 ul Hyb = 4.0pM PhiX, 0.0002N NaOH 510 | 511 | e. (4.0pM Lib x 900 ul) + (4.0pM PhiX x 100 ul) = solution loaded 512 | 513 | f. Solution loaded is 4.0pM overall with a 3.6pM Library 514 | concentration, 0.4pM PhiX concentration, and 0.000515N NaOH 515 | 516 | 6. For a 15% PhiX run, combine 850 ul of 4.0pM Library and 150 ul PhiX 517 | in a final tube. Vortex. 518 | 519 | 7. When the reagent cartridge has thawed, dry bottom with paper towel. 520 | Invert the cartridge repeatedly to check each well is thawed. This 521 | also serves to mix the reagents. Place in hood. 522 | 523 | 8. Using a clean 1000 ul pipette tip, break the foil covering wells 12, 13, 14, and 17 524 | of the reagent cartridge. Use a new tip each time. 525 | 526 | 9. Load 600 ul of the final Libary/PhiX solution into well 17 on the reagent cartridge. 527 | 528 | 10. Place 3 ul of the 100 uM Read 1 Sequencing Primer(s) into a clean 529 | PCR tube. Repeat in separate tubes for the Index Primer(s) and Read 530 | 2 Sequencing Primer(s). 531 | 532 | 11. Use an extra long 20 ul tip and pipetter to transfer the 3 ul of Read 1 Sequencing Primer 533 | to the bottom of well 12 and pipette to mix. Repeat this process spiking the Index 534 | Primer into well 13 and the Read 2 Sequencing Primer into well 14. 535 | 536 | 12. Set reagent cartridge aside. Unbox flow cell and PR2 bottle. 537 | 538 | 13. Thoroughly rinse the flow cell with Milli-Q water. Carefully dry by 539 | blotting with lint free wipes (Kimwipes). Give special attention to 540 | the edges and points of intersection between the glass and plastic. 541 | 542 | 14. Wet a new wipe with 100% alcohol and wipe the glass on both sides 543 | avoiding the rubber intake ports. 544 | 545 | 15. Visually inspect the flow cell to ensure there are no blemishes, 546 | particles, or fibers on the glass. 547 | 548 | 16. Transfer reagent cartridge, flow cell, PR2 bottle, and flash drive 549 | with the sample sheet to the MiSeq. 550 | 551 | 17. Copy Sample Sheet from the flash drive to the "Sample Sheets" folder on the desktop of the MiSeq. 552 | 553 | 18. Follow on screen instructions to load the flow cell, reagent 554 | cartridge, and PR2 bottle. Empty and replace the waste bottle. 555 | 556 | 19. Ensure the machine recognizes the correct sample sheet and the run 557 | parameters are correct. 558 | 559 | 20. Wait for the MiSeq to perform its pre-run checks, and press start. 560 | NOTE: If the pre-run check fails, try wiping down the flow cell again. 561 | 562 | 563 | ***5.8) Run Monitoring*** 564 | 565 | 1. The run should be monitored periodically using Illumina Sequence 566 | Analysis Viewer. 567 | 568 | 2. Ideal parameters for a 90% 16S run: 569 | 570 | a. Cluster density 700-800k/mm2 for v2 kits 571 | 572 | b. Cluster density 1000-1100k/mm2 for v3 kits 573 | 574 | c. \>85% clusters passing filter 575 | 576 | d. 15% aligned (amount of PhiX) 577 | 578 | e. No spikes in corrected intensity plot 579 | 580 | f. All indices identified following index reads 581 | 582 | g. Final \>Q30 score of \>70% 583 | 584 | 585 | ***5.9) Final Steps*** 586 | 587 | 1. Perform a post run wash on the MiSeq. 588 | 589 | 2. Dispose of liquid waste in appropriate hazardous jug and reagent 590 | cartridge in hazardous bucket. 591 | 592 | 3. When MiSeq Reporter finishes, go to the 'MiSeq Output' folder to transfer files as need be. 593 | 594 | 4. Perform maintenance or standby wash if required. 595 | 596 | 5. Check data to confirm they are of sufficient quality and 597 | quantity. 598 | 599 | 600 | ###Appendix A: Adding An Assay To Illumina Experiment Manager 601 | 602 | 603 | Note: You can skip steps 2-9 by simply saving the 604 | [Schloss.txt](https://github.com/SchlossLab/MiSeq_WetLab_SOP/blob/master/Schloss.txt) 605 | file to the "C:\\program files\\Illumina\\Illumina Experiment Manager\\Sample Prep Kits" directory. 606 | 607 | **Introduction** 608 | 609 | Illumina Experiment Manager (IEM) is used to generate sample plates and 610 | sheets. A new assay must be added to the system to efficiently prepare 611 | sample sheets for 16S sequencing. Not only does this eliminate the need 612 | to manually assemble a sample sheet for 16S runs, but allows the user to 613 | retain use of the IEM index analysis feature. This ensures the indices 614 | selected for a particular run have sufficient diversity on a 615 | cycle-by-cycle basis and will successfully demultiplex. 616 | 617 | Procedure 618 | 619 | 1. Open C:\\program files\\Illumina\\Illumina Experiment 620 | Manager\\Sample Prep Kits 621 | 622 | 2. Copy the file Nextera.txt, and rename the file Schloss.txt 623 | 624 | 3. Open in a text editor 625 | 626 | 4. Under [Name] change to Schloss 627 | 628 | 5. Under [PlateExtension] change to Schloss 629 | 630 | 6. Under [I7], clear the Nextera indices and paste in the SA701 through 631 | SB712 index names, and the REVERSE COMPLIMENT of the primer index 632 | sequences. 633 | 634 | 7. Under [I5], remove the Nextera indices and paste in the SA501 635 | through SB512 index names and index sequences with no alteration 636 | (NOT the reverse compliment). 637 | 638 | 8. Under [DefaultLayout\_SingleIndex] and [DefaultLayout\_DualIndex] 639 | each well of a 96 well plate is listed with a corresponding Nextera 640 | index name. These must be replaced with Schloss index names. It is 641 | recommended that a text editor with column select capability be used 642 | to leave the index name number unchanged (i.e. 701, 702, etc.), 643 | while replacing the character ”N” for Nextera with “SA” for Schloss 644 | A. 645 | 646 | 9. Save and close. 647 | 648 | 10. Return to the IEM folder and open the Applications folder. Open and 649 | edit the file GenerateFASTQ.txt. 650 | 651 | 11. Add a line at to the bottom with the text “Schloss” 652 | 653 | ###Appendix B: Creating Sample Plates and a Sample Sheet in IEM 654 | 655 | ***Part1: Creating Sample Plates*** 656 | 657 | 1. Open IEM and click Create Sample Plate. 658 | 2. Select "Schloss" from the Sample Perp Kit Selection menu. Click Next. 659 | 3. Enter the plate name (e.g. projectName_plate1). Click Next. 660 | 4. Click the Plate tab for a 96-well plate format. 661 | 5. Copy and paste sample names from an excel file, csv, etc., or enter the names manually. 662 | 6. Select the appropriate I7 and I5 indices in the upper and left pull-down menus using the following scheme. 663 | a. Plate 1: A701 – A712 with A501 – A508 664 | b. Plate 2: A701 – A712 with B501 – B508 665 | c. Plate 3: B701 – B712 with B501 – B508 666 | d. Plate 4: B701 – B712 with A501 – A508 667 | Note: You can select the first two indices, click and drag to highlight the remaing index spots, then click "fill right" or "fill down". 668 | 7. Click Finish, then Save. 669 | 8. Repeat steps 1-7 for all sample plates. 670 | 671 | ***Part2: Creating a Sample Sheet*** 672 | 673 | 1. From the IEM main menu, select Create Sample Sheet. 674 | 2. On the Instrument Selction page, select the MiSeq and click Next. 675 | 3. On the MiSeq Application Selection page, click Other, Fastq Only, Next. 676 | 4. Enter the barcode for the MiSeq Reagent Kit being used for the run. 677 | 5. Select "Schloss" as the Sample Prep Kit. 678 | 6. Enter the Experiment Name, Investigator Name, and Description. 679 | 7. Change the number of cycles to 251 for both read 1 and 2. Click Next. 680 | Note: You should not need to check or uncheck any boxes on the right. They should remain unchanged after selecting 681 | the Schloss Sample Prep Kit. 682 | 8. On the Sample Selection page, uncheck the Maximize box in the upper right corner. 683 | 9. Click Select Plate in upper left, and select the appropriate sample plate file for plate 1. 684 | 10. Click Select All at the bottom, then Add Selected Samples. 685 | 11. Check the Sample Sheet Status on the right. 686 | Note: For plate 1, IEM may give a warning of low diversity. This will go away when more plates are added. 687 | As long as the status is not "Invalid", you may proceed. 688 | 12. Repeat steps 9-11 for the remaining sample plates. 689 | 13. Click Finish, then Save. 690 | Note: In most cases the Sample Sheet should be saved to a flash drive to be transfered to the MiSeq. 691 | 692 | ###Appendix C: Primer design 693 | 694 | Overall design considerations 695 | 696 | - The sequencing primers must have a melting temperature near 65°C. 697 | This can be achieved by altering the pad sequence 698 | 699 | - The index sequences must balance the number of bases at each 700 | position. The index sequences listed here have a 25% ATGC 701 | composition at each site. If you are going to cherry pick indices 702 | from the list, make sure that you have even representation. 703 | 704 | Generic PCR primer design: 705 | 706 | AATGATACGGCGACCACCGAGATCTACAC \\\\<16Sf\> VX.N5?? 707 | 708 | CAAGCAGAAGACGGCATACGAGAT \\\\<16Sr\> VX.N7?? 709 | 710 | Generic read 1 primer design 711 | 712 | \\\<16Sf\> VX.read1 713 | 714 | Generic read 2 primer design 715 | 716 | \\\<16Sr\> VX.read2 717 | 718 | Generic index read primer design 719 | 720 | Reverse complement of (\\\<16Sr\>) VX.p7\_index 721 | 722 | The listed sequences in the generic design, above, are the adapter 723 | sequences to allow annealing of the amplicons to the flow cell. The i5 724 | and i7 sequences are the 8-nt index sequences. The pad is a 10-nt 725 | sequence to boost the sequencing primer melting temperatures. The link 726 | is a 2-nt sequence that is anti-complementary to the known sequences. 727 | The 16Sf and 16Sr are the gene specific primer sequences. Primers are 728 | purchased from IDT with no special purification. This system should work 729 | for any other region of the 16S rRNA gene or any other gene. The only 730 | thing to change would be the 16Sf/16Sr sequences and confirm that when 731 | combined with the pad sequence that the melting temperature is near 732 | 65°C. 733 | 734 | 16Sf 735 | 736 | V3: CCTACGGGAGGCAGCAG 737 | 738 | V4: GTGCCAGCMGCCGCGGTAA 739 | 740 | 16Sr 741 | 742 | V4: GGACTACHVGGGTWTCTAAT 743 | 744 | V5: CCCGTCAATTCMTTTRAGT 745 | 746 | Link: 747 | 748 | V4f: GT 749 | 750 | V4r: CC 751 | 752 | V3f: GG 753 | 754 | V5r: GG 755 | 756 | Pad: 757 | 758 | Forward: TATGGTAATT 759 | 760 | Reverse: AGTCAGTCAG 761 | 762 | i5 763 | 764 | SA501 ATCGTACG 765 | 766 | SA502 ACTATCTG 767 | 768 | SA503 TAGCGAGT 769 | 770 | SA504 CTGCGTGT 771 | 772 | SA505 TCATCGAG 773 | 774 | SA506 CGTGAGTG 775 | 776 | SA507 GGATATCT 777 | 778 | SA508 GACACCGT 779 | 780 | SB501 CTACTATA 781 | 782 | SB502 CGTTACTA 783 | 784 | SB503 AGAGTCAC 785 | 786 | SB504 TACGAGAC 787 | 788 | SB505 ACGTCTCG 789 | 790 | SB506 TCGACGAG 791 | 792 | SB507 GATCGTGT 793 | 794 | SB508 GTCAGATA 795 | 796 | SC501 ACGACGTG 797 | 798 | SC502 ATATACAC 799 | 800 | SC503 CGTCGCTA 801 | 802 | SC504 CTAGAGCT 803 | 804 | SC505 GCTCTAGT 805 | 806 | SC506 GACACTGA 807 | 808 | SC507 TGCGTACG 809 | 810 | SC508 TAGTGTAG 811 | 812 | SD501 AAGCAGCA 813 | 814 | SD502 ACGCGTGA 815 | 816 | SD503 CGATCTAC 817 | 818 | SD504 TGCGTCAC 819 | 820 | SD505 GTCTAGTG 821 | 822 | SD506 CTAGTATG 823 | 824 | SD507 GATAGCGT 825 | 826 | SD508 TCTACACT 827 | 828 | i7 829 | 830 | SA701 AACTCTCG 831 | 832 | SA702 ACTATGTC 833 | 834 | SA703 AGTAGCGT 835 | 836 | SA704 CAGTGAGT 837 | 838 | SA705 CGTACTCA 839 | 840 | SA706 CTACGCAG 841 | 842 | SA707 GGAGACTA 843 | 844 | SA708 GTCGCTCG 845 | 846 | SA709 GTCGTAGT 847 | 848 | SA710 TAGCAGAC 849 | 850 | SA711 TCATAGAC 851 | 852 | SA712 TCGCTATA 853 | 854 | SB701 AAGTCGAG 855 | 856 | SB702 ATACTTCG 857 | 858 | SB703 AGCTGCTA 859 | 860 | SB704 CATAGAGA 861 | 862 | SB705 CGTAGATC 863 | 864 | SB706 CTCGTTAC 865 | 866 | SB707 GCGCACGT 867 | 868 | SB708 GGTACTAT 869 | 870 | SB709 GTATACGC 871 | 872 | SB710 TACGAGCA 873 | 874 | SB711 TCAGCGTT 875 | 876 | SB712 TCGCTACG 877 | 878 | SC701 ACCTACTG 879 | 880 | SC702 AGCGCTAT 881 | 882 | SC703 AGTCTAGA 883 | 884 | SC704 CATGAGGA 885 | 886 | SC705 CTAGCTCG 887 | 888 | SC706 CTCTAGAG 889 | 890 | SC707 GAGCTCAT 891 | 892 | SC708 GGTATGCT 893 | 894 | SC709 GTATGACG 895 | 896 | SC710 TAGACTGA 897 | 898 | SC711 TCACGATG 899 | 900 | SC712 TCGAGCTC 901 | 902 | SD701 ACCTAGTA 903 | 904 | SD702 ACGTACGT 905 | 906 | SD703 ATATCGCG 907 | 908 | SD704 CACGATAG 909 | 910 | SD705 CGTATCGC 911 | 912 | SD706 CTGCGACT 913 | 914 | SD707 GCTGTAAC 915 | 916 | SD708 GGACGTTA 917 | 918 | SD709 GGTCGTAG 919 | 920 | SD710 TAAGTCTC 921 | 922 | SD711 TACACAGT 923 | 924 | SD712 TTGACGCA 925 | 926 | Primers used to amplify 1536 samples using the V4 region. If you only want 384 then use a subset of the listed primers (e.g. all of the v4.SA5* and v4.SB5* and v4.SA7* and v4.SB7* primers): 927 | 928 | v4.SA501 929 | AATGATACGGCGACCACCGAGATCTACACATCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 930 | 931 | v4.SA502 932 | AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 933 | 934 | v4.SA503 935 | AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 936 | 937 | v4.SA504 938 | AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 939 | 940 | v4.SA505 941 | AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 942 | 943 | v4.SA506 944 | AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 945 | 946 | v4.SA507 947 | AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 948 | 949 | v4.SA508 950 | AATGATACGGCGACCACCGAGATCTACACGACACCGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 951 | 952 | v4.SB501 953 | AATGATACGGCGACCACCGAGATCTACACCTACTATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 954 | 955 | v4.SB502 956 | AATGATACGGCGACCACCGAGATCTACACCGTTACTATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 957 | 958 | v4.SB503 959 | AATGATACGGCGACCACCGAGATCTACACAGAGTCACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 960 | 961 | v4.SB504 962 | AATGATACGGCGACCACCGAGATCTACACTACGAGACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 963 | 964 | v4.SB505 965 | AATGATACGGCGACCACCGAGATCTACACACGTCTCGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 966 | 967 | v4.SB506 968 | AATGATACGGCGACCACCGAGATCTACACTCGACGAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 969 | 970 | v4.SB507 971 | AATGATACGGCGACCACCGAGATCTACACGATCGTGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 972 | 973 | v4.SB508 974 | AATGATACGGCGACCACCGAGATCTACACGTCAGATATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 975 | 976 | v4.SC501 977 | AATGATACGGCGACCACCGAGATCTACACACGACGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 978 | 979 | v4.SC502 980 | AATGATACGGCGACCACCGAGATCTACACATATACACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 981 | 982 | v4.SC503 983 | AATGATACGGCGACCACCGAGATCTACACCGTCGCTATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 984 | 985 | v4.SC504 986 | AATGATACGGCGACCACCGAGATCTACACCTAGAGCTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 987 | 988 | v4.SC505 AATGATACGGCGACCACCGAGATCTACACGCTCTAGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 989 | 990 | v4.SC506 AATGATACGGCGACCACCGAGATCTACACGACACTGATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 991 | 992 | v4.SC507 AATGATACGGCGACCACCGAGATCTACACTGCGTACGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 993 | 994 | v4.SC508 AATGATACGGCGACCACCGAGATCTACACTAGTGTAGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 995 | 996 | v4.SD501 AATGATACGGCGACCACCGAGATCTACACAAGCAGCATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 997 | 998 | v4.SD502 AATGATACGGCGACCACCGAGATCTACACACGCGTGATATGGTAATTGTGTGCCAGCMGCCGCGGTAA 999 | 1000 | v4.SD503 AATGATACGGCGACCACCGAGATCTACACCGATCTACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1001 | 1002 | v4.SD504 AATGATACGGCGACCACCGAGATCTACACTGCGTCACTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1003 | 1004 | v4.SD505 AATGATACGGCGACCACCGAGATCTACACGTCTAGTGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1005 | 1006 | v4.SD506 AATGATACGGCGACCACCGAGATCTACACCTAGTATGTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1007 | 1008 | v4.SD507 AATGATACGGCGACCACCGAGATCTACACGATAGCGTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1009 | 1010 | v4.SD508 AATGATACGGCGACCACCGAGATCTACACTCTACACTTATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1011 | 1012 | v4.SA701 CAAGCAGAAGACGGCATACGAGATAACTCTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1013 | 1014 | v4.SA702 CAAGCAGAAGACGGCATACGAGATACTATGTCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1015 | 1016 | v4.SA703 CAAGCAGAAGACGGCATACGAGATAGTAGCGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1017 | 1018 | v4.SA704 CAAGCAGAAGACGGCATACGAGATCAGTGAGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1019 | 1020 | v4.SA705 CAAGCAGAAGACGGCATACGAGATCGTACTCAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1021 | 1022 | v4.SA706 CAAGCAGAAGACGGCATACGAGATCTACGCAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1023 | 1024 | v4.SA707 1025 | CAAGCAGAAGACGGCATACGAGATGGAGACTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1026 | 1027 | v4.SA708 1028 | CAAGCAGAAGACGGCATACGAGATGTCGCTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1029 | 1030 | v4.SA709 1031 | CAAGCAGAAGACGGCATACGAGATGTCGTAGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1032 | 1033 | v4.SA710 1034 | CAAGCAGAAGACGGCATACGAGATTAGCAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1035 | 1036 | v4.SA711 1037 | CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1038 | 1039 | v4.SA712 1040 | CAAGCAGAAGACGGCATACGAGATTCGCTATAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1041 | 1042 | v4.SB701 1043 | CAAGCAGAAGACGGCATACGAGATAAGTCGAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1044 | 1045 | v4.SB702 1046 | CAAGCAGAAGACGGCATACGAGATATACTTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1047 | 1048 | v4.SB703 1049 | CAAGCAGAAGACGGCATACGAGATAGCTGCTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1050 | 1051 | v4.SB704 1052 | CAAGCAGAAGACGGCATACGAGATCATAGAGAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1053 | 1054 | v4.SB705 1055 | CAAGCAGAAGACGGCATACGAGATCGTAGATCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1056 | 1057 | v4.SB706 1058 | CAAGCAGAAGACGGCATACGAGATCTCGTTACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1059 | 1060 | v4.SB707 1061 | CAAGCAGAAGACGGCATACGAGATGCGCACGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1062 | 1063 | v4.SB708 1064 | CAAGCAGAAGACGGCATACGAGATGGTACTATAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1065 | 1066 | v4.SB709 1067 | CAAGCAGAAGACGGCATACGAGATGTATACGCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1068 | 1069 | v4.SB710 1070 | CAAGCAGAAGACGGCATACGAGATTACGAGCAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1071 | 1072 | v4.SB711 1073 | CAAGCAGAAGACGGCATACGAGATTCAGCGTTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1074 | 1075 | v4.SB712 1076 | CAAGCAGAAGACGGCATACGAGATTCGCTACGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1077 | 1078 | v4.SC701 CAAGCAGAAGACGGCATACGAGATACCTACTGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1079 | 1080 | v4.SC702 CAAGCAGAAGACGGCATACGAGATAGCGCTATAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1081 | 1082 | v4.SC703 CAAGCAGAAGACGGCATACGAGATAGTCTAGAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1083 | 1084 | v4.SC704 CAAGCAGAAGACGGCATACGAGATCATGAGGAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1085 | 1086 | v4.SC705 CAAGCAGAAGACGGCATACGAGATCTAGCTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1087 | 1088 | v4.SC706 CAAGCAGAAGACGGCATACGAGATCTCTAGAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1089 | 1090 | v4.SC707 CAAGCAGAAGACGGCATACGAGATGAGCTCATAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1091 | 1092 | v4.SC708 CAAGCAGAAGACGGCATACGAGATGGTATGCTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1093 | 1094 | v4.SC709 CAAGCAGAAGACGGCATACGAGATGTATGACGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1095 | 1096 | v4.SC710 CAAGCAGAAGACGGCATACGAGATTAGACTGAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1097 | 1098 | v4.SC711 CAAGCAGAAGACGGCATACGAGATTCACGATGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1099 | 1100 | v4.SC712 CAAGCAGAAGACGGCATACGAGATTCGAGCTCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1101 | 1102 | v4.SD701 CAAGCAGAAGACGGCATACGAGATACCTAGTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1103 | 1104 | v4.SD702 CAAGCAGAAGACGGCATACGAGATACGTACGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1105 | 1106 | v4.SD703 CAAGCAGAAGACGGCATACGAGATATATCGCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1107 | 1108 | v4.SD704 CAAGCAGAAGACGGCATACGAGATCACGATAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1109 | 1110 | v4.SD705 CAAGCAGAAGACGGCATACGAGATCGTATCGCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1111 | 1112 | v4.SD706 CAAGCAGAAGACGGCATACGAGATCTGCGACTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1113 | 1114 | v4.SD707 CAAGCAGAAGACGGCATACGAGATGCTGTAACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1115 | 1116 | v4.SD708 CAAGCAGAAGACGGCATACGAGATGGACGTTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1117 | 1118 | v4.SD709 CAAGCAGAAGACGGCATACGAGATGGTCGTAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1119 | 1120 | v4.SD710 CAAGCAGAAGACGGCATACGAGATTAAGTCTCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1121 | 1122 | v4.SD711 CAAGCAGAAGACGGCATACGAGATTACACAGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1123 | 1124 | v4.SD712 CAAGCAGAAGACGGCATACGAGATTTGACGCAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1125 | 1126 | 1127 | 1128 | Read 1 primer for V4 region 1129 | 1130 | TATGGTAATTGTGTGCCAGCMGCCGCGGTAA 1131 | 1132 | Read 2 primer for V4 region 1133 | 1134 | AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 1135 | 1136 | Index primer for V4 region 1137 | 1138 | ATTAGAWACCCBDGTAGTCCGGCTGACTGACT 1139 | 1140 | [^1]: For extremely low concentration libraries we have adapted a method 1141 | published by Quail et al. 1142 | [http://www.nature.com/nmeth/journal/v5/n12/full/nmeth.1270.html](../customXml/item1.xml) 1143 | 1144 | Libraries as low as 0.1nM do not allow for sufficient dilution to 1145 | reduce NaOH to 0.001N. It must be neutralized using 200mM Tris pH 1146 | 7.0. Example: 40 ul 0.1nM library + 40 ul 0.1N NaOH. Incubate for 5 1147 | min. Add 80 ul 200mM Tris. Then add 840 ul HT1. This results in a 1148 | 4.0pM library. 1149 | -------------------------------------------------------------------------------- /Schloss.txt: -------------------------------------------------------------------------------- 1 | [Version] 2 | 1 3 | [Name] 4 | Schloss 5 | [PlateExtension] 6 | schloss 7 | [Settings] 8 | 9 | Adapter CTGTCTCTTATACACATCT 10 | ManifestExtension AmpliconManifest 11 | [I7] 12 | SA701 CGAGAGTT 13 | SA702 GACATAGT 14 | SA703 ACGCTACT 15 | SA704 ACTCACTG 16 | SA705 TGAGTACG 17 | SA706 CTGCGTAG 18 | SA707 TAGTCTCC 19 | SA708 CGAGCGAC 20 | SA709 ACTACGAC 21 | SA710 GTCTGCTA 22 | SA711 GTCTATGA 23 | SA712 TATAGCGA 24 | SB701 CTCGACTT 25 | SB702 CGAAGTAT 26 | SB703 TAGCAGCT 27 | SB704 TCTCTATG 28 | SB705 GATCTACG 29 | SB706 GTAACGAG 30 | SB707 ACGTGCGC 31 | SB708 ATAGTACC 32 | SB709 GCGTATAC 33 | SB710 TGCTCGTA 34 | SB711 AACGCTGA 35 | SB712 CGTAGCGA 36 | 37 | [I5] 38 | SA501 ATCGTACG 39 | SA502 ACTATCTG 40 | SA503 TAGCGAGT 41 | SA504 CTGCGTGT 42 | SA505 TCATCGAG 43 | SA506 CGTGAGTG 44 | SA507 GGATATCT 45 | SA508 GACACCGT 46 | SB501 CTACTATA 47 | SB502 CGTTACTA 48 | SB503 AGAGTCAC 49 | SB504 TACGAGAC 50 | SB505 ACGTCTCG 51 | SB506 TCGACGAG 52 | SB507 GATCGTGT 53 | SB508 GTCAGATA 54 | 55 | [DefaultLayout_SingleIndex] 56 | A01 SA701 57 | A02 SA702 58 | A03 SA703 59 | A04 SA704 60 | A05 SA705 61 | A06 SA706 62 | A07 SA707 63 | A08 SA708 64 | A09 SA709 65 | A10 SA710 66 | A11 SA711 67 | A12 SA712 68 | B01 SA701 69 | B02 SA702 70 | B03 SA703 71 | B04 SA704 72 | B05 SA705 73 | B06 SA706 74 | B07 SA707 75 | B08 SA708 76 | B09 SA709 77 | B10 SA710 78 | B11 SA711 79 | B12 SA712 80 | C01 SA701 81 | C02 SA702 82 | C03 SA703 83 | C04 SA704 84 | C05 SA705 85 | C06 SA706 86 | C07 SA707 87 | C08 SA708 88 | C09 SA709 89 | C10 SA710 90 | C11 SA711 91 | C12 SA712 92 | D01 SA701 93 | D02 SA702 94 | D03 SA703 95 | D04 SA704 96 | D05 SA705 97 | D06 SA706 98 | D07 SA707 99 | D08 SA708 100 | D09 SA709 101 | D10 SA710 102 | D11 SA711 103 | D12 SA712 104 | E01 SA701 105 | E02 SA702 106 | E03 SA703 107 | E04 SA704 108 | E05 SA705 109 | E06 SA706 110 | E07 SA707 111 | E08 SA708 112 | E09 SA709 113 | E10 SA710 114 | E11 SA711 115 | E12 SA712 116 | F01 SA701 117 | F02 SA702 118 | F03 SA703 119 | F04 SA704 120 | F05 SA705 121 | F06 SA706 122 | F07 SA707 123 | F08 SA708 124 | F09 SA709 125 | F10 SA710 126 | F11 SA711 127 | F12 SA712 128 | G01 SA701 129 | G02 SA702 130 | G03 NA703 131 | G04 SA704 132 | G05 SA705 133 | G06 SA706 134 | G07 SA707 135 | G08 SA708 136 | G09 SA709 137 | G10 SA710 138 | G11 SA711 139 | G12 SA712 140 | H01 SA701 141 | H02 SA702 142 | H03 SA703 143 | H04 SA704 144 | H05 SA705 145 | H06 SA706 146 | H07 SA707 147 | H08 SA708 148 | H09 SA709 149 | H10 SA710 150 | H11 SA711 151 | H12 SA712 152 | [DefaultLayout_DualIndex] 153 | A01 SA701 SA501 154 | A02 SA702 SA501 155 | A03 SA703 SA501 156 | A04 SA704 SA501 157 | A05 SA705 SA501 158 | A06 SA706 SA501 159 | A07 SA707 SA501 160 | A08 SA708 SA501 161 | A09 SA709 SA501 162 | A10 SA710 SA501 163 | A11 SA711 SA501 164 | A12 SA712 SA501 165 | B01 SA701 SA502 166 | B02 SA702 SA502 167 | B03 SA703 SA502 168 | B04 SA704 SA502 169 | B05 SA705 SA502 170 | B06 SA706 SA502 171 | B07 SA707 SA502 172 | B08 SA708 SA502 173 | B09 SA709 SA502 174 | B10 SA710 SA502 175 | B11 SA711 SA502 176 | B12 SA712 SA502 177 | C01 SA701 SA503 178 | C02 SA702 SA503 179 | C03 SA703 SA503 180 | C04 SA704 SA503 181 | C05 SA705 SA503 182 | C06 SA706 SA503 183 | C07 SA707 SA503 184 | C08 SA708 SA503 185 | C09 SA709 SA503 186 | C10 SA710 SA503 187 | C11 SA711 SA503 188 | C12 SA712 SA503 189 | D01 SA701 SA504 190 | D02 SA702 SA504 191 | D03 SA703 SA504 192 | D04 SA704 SA504 193 | D05 SA705 SA504 194 | D06 SA706 SA504 195 | D07 SA707 SA504 196 | D08 SA708 SA504 197 | D09 SA709 SA504 198 | D10 SA710 SA504 199 | D11 SA711 SA504 200 | D12 SA712 SA504 201 | E01 SA701 SA505 202 | E02 SA702 SA505 203 | E03 SA703 SA505 204 | E04 SA704 SA505 205 | E05 SA705 SA505 206 | E06 SA706 SA505 207 | E07 SA707 SA505 208 | E08 SA708 SA505 209 | E09 SA709 SA505 210 | E10 SA710 SA505 211 | E11 SA711 SA505 212 | E12 SA712 SA505 213 | F01 SA701 SA506 214 | F02 SA702 SA506 215 | F03 SA703 SA506 216 | F04 SA704 SA506 217 | F05 SA705 SA506 218 | F06 SA706 SA506 219 | F07 SA707 SA506 220 | F08 SA708 SA506 221 | F09 SA709 SA506 222 | F10 SA710 SA506 223 | F11 SA711 SA506 224 | F12 SA712 SA506 225 | G01 SA701 SA507 226 | G02 SA702 SA507 227 | G03 SA703 SA507 228 | G04 SA704 SA507 229 | G05 SA705 SA507 230 | G06 SA706 SA507 231 | G07 SA707 SA507 232 | G08 SA708 SA507 233 | G09 SA709 SA507 234 | G10 SA710 SA507 235 | G11 SA711 SA507 236 | G12 SA712 SA507 237 | H01 SA701 SA508 238 | H02 SA702 SA508 239 | H03 SA703 SA508 240 | H04 SA704 SA508 241 | H05 SA705 SA508 242 | H06 SA706 SA508 243 | H07 SA707 SA508 244 | H08 SA708 SA508 245 | H09 SA709 SA508 246 | H10 SA710 SA508 247 | H11 SA711 SA508 248 | H12 SA712 SA508 --------------------------------------------------------------------------------