(r3) RareKappaNgsPrep < Lab

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This is a protocol based on Kappa LTP Preparation Kit manual KR0453 - v3.13<br>
The main difference centers around the use of 1/2 volume in each reaction so as to get twice as many sample preps for the same price. <br>
This was validated on 2 extremely similar _clonal_ samples from Gabo by DED in July of 2014, and the final data was indistinguishable between the 1x volume sample and the 0.5x volume sample.  <br>
This has subsequently been used in more than a dozen (4-15-15) other preps (rare variant and clonal) without incident. <br>
This protocol is for rare variant applications. It is a waste of time, money, and reagents to use this protocol on samples which you do not expect and/or are not interested in mutations that exist at a frequency of <1%. Consider which protocol is best before proceeding. Please [[KappaNgsPrep][click here]] for standard protocol. <br>

---++ Overview
   1. Important Notes
   1. Anealing Adapters
   1. Harvest DNA and determine concentration.
   1. Fragment DNA.
   1. End Repair DNA.
   1. Wash
   1. dA tail 3' end of DNA fragments
   1. Wash
   1. Ligate internal barcoded adapters on
   1. Wash 
   1. Size select 
   1. PCR amplify library
   1. Wash
   1. QC
   1. Submit for sequencing

---++ Before Beginning and Important Notes
   1. Low retention tubes and filter tips should be used at __every__ step. Regular tips commonly used for !EtOH removal during wash steps.
   1. Before starting make sure ligated adapters are available in cardboard box labeled 'Annealed adapters'.
      %BR% %X% Importance of this box/these tubes never reaching RT cannot be underscored enough
   1. WFI water should be used for all steps where water is needed. Aliquots of water should be taken from bottle to avoid contamination.
   1. A 2 barcode system is currently being employed. There are 6 annealed adapters corresponding to 1 of 6 internal barcodes (IBC). External barcode(s) (EBC) are added to samples in the PCR step. Identical EBC can and should be pooled together to meet the sequencing core's minimum requirement of 10million reads per sample. __NEVER__ pool identical IBC as this type of pooling can not be reassigned at the data step. 
   1. Sample should never be vortexed, always thoroughly mixed by pipetting. This is done to avoid sample getting near the top of the tube and increasing contamination risk during magnetic bead clean up.
   1. "Freshly prepared 80% !EtOH" for cleanup reactions should be prepared daily, and be made combining 100% !EtOH with WFI water.
   1. Several different alternative options are available, steps which have alternative strategies are marked as such.  

---++ Adapter Annealation
   %T% Note, this step is only necessary if 'Annealed Adapters' tube is empty. This should be a rare step.
   %T% This step can be scaled up, but some evidence that after ~14-18 months annealed extended adapters don't function as well, so don't make too much excess.
   1 Mix the following in a PCR tube:
      * 20µl of Adapter_forward (100pmol/µl)
      * 20µl of MI_Reverse (100pmol/µl)
   1 Use thermocycler with following conditions to anneal the 2 primers together:
      1. 97C   2minutes
      1. 97C   1minute
         1. Repeat previous step for 72 total cycles at -1C per cycle
      1. 25C   5 minutes
      %BR% After this step, adapters should __NEVER__ be warmed above 25C.
   1 To the annealed adapters, add the following extension reaction reagents:
|Item| Amount|
|WFI water| 5.5µl|
|10x NEB 2 Buffer| 6µl|
|2.5mM dNTP| 6µl|
|Klenow exo<sup>-</sup> (5 units/µl)| 2.5µl|
   1 Incubate 1 hr at 37°C in thermocycler.
   1 Ethanol precipitate by adding the following. Note the times and spins of this precipitation are excessive, but they are an attempt to increase recovery as the adapters are quite expensive:
|Item| Amount|
|Glycogen (20µg/µl)| 2µl|
|!NaOAC (3M pH 5.2)| 6.2µl|
|100% !EtOH| 170.5µl|
   1 Mix well and precipitate overnight at -20°C
   1 Spin at max speed for 30 minutes at 4°C.
   1 Decant carefully and/or pipette without disturbing pellet.
   1 Add 1mL 70% !EtOH and resuspend via flicking tube.
   1 Spin at max speed for 30 minutes at 4°C.
   1 Decant carefully and/or pipette without disturbing pellet.
   1 Air dry 10 minutes inverted sitting on fresh kimwipe.
   1 Air dry 5 minutes upright near burning flame.
   1 Resuspend in 40µl of WFI water.
   1 dA tail the adapters by adding the following:
|Item| Amount|
|WFI water| 5.5µl|
|10x NEB 2 Buffer| 6µl|
|10mM dATP| 6µl|
|Klenow exo<sup>-</sup> (5 units/µl)| 2.5µl|
   1 Incubate 1 hr at 37°C in thermocycler.
   1 Ethanol precipitate by adding the following. Note the times and spins of this precipitation are excessive, but they are an attempt to increase recovery as the adapters are quite expensive:
|Item| Amount|
|Glycogen (20µg/µl)| 2µl|
|!NaOAC (3M pH 5.2)| 6.2µl|
|100% !EtOH| 170.5µl|
   1 Mix well and precipitate overnight at -20°C
   1 Spin at max speed for 30 minutes at 4°C.
   1 Decant carefully and/or pipette without disturbing pellet.
   1 Add 1mL 70% !EtOH and resuspend via flicking tube.
   1 Spin at max speed for 30 minutes at 4°C.
   1 Decant carefully and/or pipette without disturbing pellet.
   1 Air dry 10 minutes inverted sitting on fresh kimwipe.
   1 Air dry 5 minutes upright near burning flame.
   1 Resuspend in 40µl of WFI water.
   1 Make sure tube is well labeled.
   1 Store -20°C

---++ Harvest DNA and determine Concentration
   1. Harvest DNA from sample using Invitrogen !PureLink Genomic DNA kit using standard protocol.
   1. It is important to elute in elution buffer rather than water.
   1. Use Qbit to determine concentration.

---++ Fragment DNA
   * 2 different shearing protocols have been developed, choose appropriate based on cost and time. Very likely covaris method falling out of favor due to 5$ per tube reagent cost for single shearing event.
   * The goal of this step is to transfer 500ng to 1µg of sheared DNA in 25µl to a low retention eppendorf tube.

---+++ Covaris shearing
   * Successfully done with 500ng of sheared DNA in 25µl. 
      %BR% %X% During ligation 0.4µl of adapter should be used, __NOT__ 1µl using 500ng in the library prep
   1. Place between 1µg and 4 µg of DNA in a covaris microtube (total µg can be increased to 10, but should never be necessary, or decreased below 1µg if necessary).
   1. Bring total volume up to 50 __or__ 130µl using Invitrogen Elution Buffer.
      * Covaris only recommends 130 and 50 µl with the microtubes as other volumes can create air bubbles and lead to poor shearing.
   1. Signup for an appropriate amount of time in the GSAF core, and take all samples to core.
   1. Use appropriate shearing conditions for the Covaris instrument either from Barrick lab folder, or GSAF folder (if GSAF protocol used, save copy to Barrick folder).
      1. For standard sequencing projects, ~300bp is a good target size (if other size used adapter concentrations need to be adjusted).
      1. Consult Covaris manual for other size distributions.
   1. Spin tubes down in black plates on plate centrifuge (plates have shallow wells that cause the bottom of the Covaris tube to reach the bottom of the plate so there is no force on the lid)
   1. Transfer 1µg of sheared DNA to low retention eppendorf tube.
   1. Adjust volume to 25 µl using speedvac if necessary.
   1. Volume must be 25 µl, different amounts of DNA can be used with subsequent adjustments to adapter amounts. If attempted, update appropriate sections with working concentrations.
   1. Store remaining sheared DNA at -20C at a minimum until sequencing data is back, it can be used to save a Covaris tube if sample fails, or for other experiments.

---+++ NEB Fragmentase
   * Protocol employed by Mike due to large quantity of samples. If no details listed here, [[KappaNgsPrep][check standard protocol]] or see him for details if neither contains details.

---++ End Repair DNA
   1. Move blue heat block to -4C, and set to 20C before starting so heat block will adjust temperature accordingly.
   1. Place Ampure beads on benchtop to warm to room temperature.
   1. To each sample tube add:
|Item| Amount|
|WFI water| 4µl|
|10x KAPA End Repair Buffer| 3.5µl|
|KAPA End Repair Enzyme Mix| 2.5µl|
   1. Pipette to mix.
   1. Incubate 20C for 30 minutes
   1. Remove tubes from block, increase temperature to 30C.

---++ Wash
   1. To each 35µl reaction, add 60µl of room temperature Ampure beads.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully remove and discard supernatant without disturbing the beads.
   1. Add 200 µl of 80% !EtOH.
   1. Allow to incubate more than 30 seconds at room temperature. It is not necessary to resuspend the beads.
   1. Repeat for a 2nd wash.
   1. Allow beads to dry at room temperature for all ethanol to evaporate.
      1. ethanol carryover will interfere with subsequent steps, and over-drying of beads (typically characterized by cracks appearing in the beads)
   1. Remove beads from magnet.

---++ T-Tailing Reaction
   1. %X% This step uses __NOTHING__ from the kapa kit besides the PEG/NaCL solution.
   1. Make sure heat block is set to 30C and holding.
   1. Pull PEG/NaCL SPRI Solution and place on bench to warm to room temperature
   1. To each tube add the following:
|Item| Amount|
|WFI water| 19.5µl|
|10x NEB buffer 2| 2.5µl|
|10mM dTTP| 2.5µl|
|Klenow exo- (5units/µl)| 0.5µl|
   1. Mix by pipetting till homogeneous solution
   1. Incubate 30 minutes at 30C.
   1. Remove tubes from block.
   1. Remove block from base, and place on bench in cold room. Set block to 20C. Check back periodically (at least 15 minutes needed) for block to stay at or below 20C once back in base. Ice buckets can be used instead of cold room bench for better heat dispersal.

---++ Wash
   1. To each 25µl reaction, add 45µl PEG/NaCl Solution.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully remove and discard supernatant without disturbing the beads.
   1. Add 200 µl of 80% !EtOH.
   1. Allow to incubate more than 30 seconds at room temperature. It is not necessary to resuspend the beads.
   1. Repeat for a 2nd wash.
   1. Allow beads to dry at room temperature for all ethanol to evaporate.
      1. ethanol carryover will interfere with subsequent steps, and over-drying of beads (typically characterized by cracks appearing in the beads)
   1. Remove beads from magnet.

---++ Adapter Ligation
   1. Make sure heat block is holding at 20C.
   1. Protocol assumes ~300bp shearing, if different sized inserts, adjust Adapter amounts.
   1. Pull Ampure beads from 4C and set on bench to warm to room temperature.
   1. To each tube add the following:
|Item| Amount|
|WFI water| 16µl|
|5x KAPA Ligation Buffer| 5µl|
|KAPA t4 DNA Ligase| 2.5µl|
|Adapter (__NOT IN Master Mix__)| 1.5µl|
   1. Mix by pipetting to homogeneous mixture. 
   1. Incubate 20C for 30 minutes.
   1. Remove tubes from heat block, turn heat block off, bring back into lab.

---++ Wash
   1. The ligation buffer interferes with the normal precipitation of DNA, therefore must be washed before size selection.
   1. To each 25µl reaction, add 25µl PEG/NaCl Solution.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully remove and discard supernatant without disturbing the beads.
   1. Add 200 µl of 80% !EtOH.
   1. Allow to incubate more than 30 seconds at room temperature. It is not necessary to resuspend the beads.
   1. Repeat for a 2nd wash.
   1. Allow beads to dry at room temperature for all ethanol to evaporate.
      1. ethanol carryover will interfere with subsequent steps, and over-drying of beads (typically characterized by cracks appearing in the beads)
   1. Remove beads from magnet.
   1. Resuspend in 100µl of WFI water.

---++ Size Selection
   1. To each 100µl reaction, add 60µl PEG/NaCl Solution.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
      * This will bind DNA fragments of ~450bp
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully transfer ~155µl of supernatant to a new tube. It is critical that no beads be transfered with supernatant.
   1. To each new tube containing 155µl of DNA smaller than 450bp add 20µl of Ampure beads.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully remove and discard supernatant without disturbing the beads.
   1. Add 200 µl of 80% !EtOH.
   1. Allow to incubate more than 30 seconds at room temperature. It is not necessary to resuspend the beads.
   1. Repeat for a 2nd wash.
   1. Allow beads to dry at room temperature for all ethanol to evaporate.
      1. ethanol carryover will interfere with subsequent steps, and over-drying of beads (typically characterized by cracks appearing in the beads)
   1. Remove beads from magnet.
   1. Resuspend in 50µl of WFI water.

---++ PCR Addition of EBC
   * Take note of which external barcode is being added on this step for the purpose of pooling multiple samples.
   1. Pull Ampure beads to allow to warm to room temperature.
   1. Combine the following in a PCR tube:
|Item|  Amount|
|Size Selected DNA| 10 µl|
|EBC Barcode mix (10µM)| 2.5 µl|
|2x KAPA !HiFi !HotStart Ready Mix| 12.5 µl|

   * Store remaining DNA at -20C in labeled tube. This will be used if the PCR is unsuccessful, or if further optimization is required.

|   *PCR Conditions*   |||
|Step|Temp|Time|
|1|  98  |  45 sec  |
|2|  98  |  15 sec  |
|3|  60  |  30 sec  |
|4|  72  |  30 sec  |
|5|  go to  |  Step 2  |
|6|  72  |  1 min  |

   * Minimum number of cycles should be preformed to maximize diversity. This is esspecially important for mixed population sequencings. Typically 5-7 Cycles is sufficient.

---++ Wash
   1. To each 25µl reaction, add 25µl of room temperature Ampure beads.
   1. Mix by pipetting up and down till homogeneous mixture. 
   1. Incubate at room temperature for 10 minutes for DNA to precipitate onto beads.
   1. Place tubes on magnet until liquid is clear (usually ~5 minutes).
   1. Carefully remove and discard supernatant without disturbing the beads.
   1. Add 200 µl of 80% !EtOH.
   1. Allow to incubate more than 30 seconds at room temperature. It is not necessary to resuspend the beads.
   1. Repeat for a 2nd wash.
   1. Allow beads to dry at room temperature for all ethanol to evaporate.
      1. ethanol carryover will interfere with subsequent steps, and over-drying of beads (typically characterized by cracks appearing in the beads)
   1. Remove beads from magnet.
   1. Resuspend in 25µl WFI water or TE.

---++ QC
   1. Use 1 µl of sample for tape station.
      * If substantial dimer contamination, rewash, possibly adding slightly less PEG/NaCl solution than a 1:1 ratio.
      * If product not visible, toubleshoot PCR step first, possibly by increasing 2-3 cycles as this is the cheapest and most likely failure step.
   1. Use 2 µl for Qbit.
   1. Pool samples with different IBC and identical EBC into a single submission tube for Core. This should be done based either on the Qbit readings, or on the fraction of correctly sized product from the tape station if different amounts of Adapter contamination detected (requires that correctly sized peak be significantly greater than the off product). 
   1. Create job [[https://wikis.utexas.edu/display/GSAF/Requesting+new+sequencing+job][at the GSAF website]]
      * only list the external barcode in communications with the core. 
   1. When job approved, take sample to GSAF core.
   1. For more information about data retrieval and analysis visit internal site InternalNgsData (login required). 

   


-- Main.DanielDeatherage - 29 Apr 2015
Barrick Lab > KappaNgsPrep > RareKappaNgsPrep
Topic revision: r3 - 2015-04-29 - 21:37:28 - Main.DanielDeatherage