===== Dissection, Extraction, and Library Preparation ======
**Guest Lecturer:  Steven Weber**\\
Full Presentation: {{lecture_notes:slug_presentation.pdf}}

==== General Notes ====
  - Administrative: read about your [[teams: | team's]] assigned assembler. 
  - Our data is from one wild-caught slug (//Ariolimax dolichophallus//). 
  - Library prep is not a nice clean process, every possible weird thing that can happen with the DNA pretty much does. 
  - What data do we have? 
    - Full lane of larger insert library on Miseq (2x300 bp, ~26M reads)
    - Full Hiseq lanes of each library (2x100 bp, ~150M reads each)
    - Mate pair library has been sequenced, need an assembly of shotgun data first (to map the mate pair reads onto) to determine quality of mate pair library reads. 

==== A. Dissection ====
  - Received frozen slug, thawed in 37°C water bath
  - Made careful incision on side of slug, being careful not to break intestinal tissue
  - Isolated and identified various body parts
  - Took out pieces of all major organs/body parts and put them into separate tubes
  - Stored at -80°C

==== B. Extraction (using Omega E.Z.N.A. Mollusk DNA kit) ====

  - Took pieces of the digestive gland, albumen gland and kidney and weighed them (25mg kidney, 120mg albumen, 60mg digestive gland)
  - Placed a marble mortar and pestle into a bed of dry ice
  - Poured liquid nitrogen into mortar and placed sample into it, letting it freeze completely
  - Ground sample to fine powder as the liquid nitrogen boiled off
  - Since the mortar is already cold, sample will remain in powder form even when all of the liquid nitrogen boils off
  - Scooped powdered sample into tube containing buffer ML1 and Proteinase K, and incubated at 60°C until the sample was fully solubilized (Note: albumen and digestive gland were split into two tubes/columns due to mass of sample)
  - Added chloroform:isoamyl alcohol (24:1), centrifuged at 10,000 x g for 2 minutes. This step removes the mucopolysaccharides from our sample, and leaves an upper aqueous phase containing our soluble DNA.
  - We carefully pipetted the aqueous phase into a clean 1.5mL microcentrifuge tube and added buffer MBL and RNase A, and incubated at 70°C for 10 minutes.
  - Once the sample cooled to room temperature we added absolute ethanol
  - Transferred 750ul of each sample to the HiBind® DNA Mini Columns
  - Spun columns at 10,000 x g for 1 minute, discarded filtrate
  - Repeated steps 10 and 11 until all of sample was applied to columns
  - Added 500ul HBC buffer with isopropanol, spun at 10,000 x g for 30sec, discarded filtrate
  - Added 700ul DNA Wash Buffer with ethanol, spun at 10,000 x g for 1 minute, discarded filtrate
  - Repeated steps 13 and 14 once
  - Dry spun for 2 minutes at maximum speed (14,000 x g)
  - Transferred columns to clean 1.5ul microcentrifuge tubes
  - Added 100uL Elution Buffer (preheated to 70°C), let sit at room temperature for 2 minutes, spun at 10,000 x g for 1 minute
  - Stored DNA at 4°C

==== C. Illumina Library Prep ====

(Two library preps side by side in two different size ranges)

  -   Sheared DNA to two different sizes, ~400bp and ~600bp (6ug into each).	
  -   Size selected for two ranges, 350-500bp and 500-700bp.
  -   Blunt End Repair with 50ul of sample plus the following: 7.12ul ddH2O, 7ul Buffer Tango (10x), 0.28ul dNTPs (25mM each), 0.7ul  ATP (100mM), 3.5ul PNK (10U/ul), 1.4ul T4 DNA Polymerase (5U/ul). Incubated at 25°C for 15 minutes then at 12°C for 5 minutes. Cleaned up reaction with 1.8x 18% PEG SPRI beads. Washed 2x with 80% EtOH. Eluted DNA in 20ul TE. Size selected, blunt ended DNA amounts: 1,252ng (350-500bp: 62.6ng/ul x 20ul) and 400ng (500-700bp: 20.0ng/ul x 20ul)
  -   Adapter Ligation with 20ul sample plus the following: 10ul ddH2O, 4ul T4 Ligase Buffer + 10mM ATP (10x), 4ul PEG-4000 (50%), 1ul P5+P7 adapters (100uM each), 1ul T4 DNA Ligase (5U/ul). Incubated at 22°C for 30 minutes. Cleaned up reaction with 1.8x 18% PEG SPRI beads (same as last time).
  -   Adapter fill-in with 20ul sample plus the following: 14.1ul ddH2O, 4ul ThermoPol Buffer (10x), 0.4ul dNTPs (25uM each), 1.5ul Bst DNA Polymerase (8U/ul). Incubated at 37°C for 20 minutes. Cleaned up reaction with 1.5x 18% PEG SPRI beads. Washed 2x with 80% EtOH. Eluted DNA in 30ul TE. Measure concentration by Qubit: 840ng (300-550bp: 28ng/ul x 30ul) and 354ng (550-750bp: 11.8ng/ul x 30ul)
  - 	Indexing PCR (index 89 for 300-550bp, index 90 for 550-750bp). Performed 10 cycles of PCR with 200ng of each sample (2 rxns each with 100ng each): 25ul KAPA 2x Polymerase mix, 2.5ul sample, 1ul Primer IS4 (10uM), 1ul indexing primer (10uM), 20.5ul ddH2O. Thermal Cycler Parameters: 98°C for 3 minutes, 98°C for 20 seconds, 65°C for 30 seconds, 72°C for 40 seconds, return to step 2 for 11 more times, 72°C for 3 minutes, 4°C forever. Measured by Qubit: 1076ng (350-500bp: 53.8ng/ul x 20ul) and 1208ng (500-700bp: 60.4ng/ul x 20ul)
  - Size selection of PCR product. Used E-gel SizeSelect 2% gel, selected for DNA in corresponding size ranges. Measured by Qubit 88.8ng (350-500bp: 4.44ng/ul x 20ul) and 147.2ng (500-700bp: 7.36ng/ul x 20ul).

==== D. Lucigen Nxseq Long Mate Pair Library Prep ====

  - Followed instructions exactly, with the exception of manually size selecting on 0.75% Agarose field inversion gel instead of selecting using AMPure beads
  - Mate pair library: essentially use a long insert size, but remove the middle so that Illumina can handle the sequencing. This helps with placing contigs together. ([[http://www.illumina.com/technology/next-generation-sequencing/mate-pair-sequencing_assay.html | more info]])