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lecture_notes:04-03-2015

Dissection, Extraction, and Library Preparation

Guest Lecturer: Steven Weber
Full Presentation: slug_presentation.pdf

General Notes

  1. Administrative: read about your team's assigned assembler.
  2. Our data is from one wild-caught slug (Ariolimax dolichophallus).
  3. Library prep is not a nice clean process, every possible weird thing that can happen with the DNA pretty much does.
  4. What data do we have?
    1. Full lane of larger insert library on Miseq (2×300 bp, ~26M reads)
    2. Full Hiseq lanes of each library (2×100 bp, ~150M reads each)
    3. 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

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

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

  1. Took pieces of the digestive gland, albumen gland and kidney and weighed them (25mg kidney, 120mg albumen, 60mg digestive gland)
  2. Placed a marble mortar and pestle into a bed of dry ice
  3. Poured liquid nitrogen into mortar and placed sample into it, letting it freeze completely
  4. Ground sample to fine powder as the liquid nitrogen boiled off
  5. Since the mortar is already cold, sample will remain in powder form even when all of the liquid nitrogen boils off
  6. 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)
  7. 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.
  8. 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.
  9. Once the sample cooled to room temperature we added absolute ethanol
  10. Transferred 750ul of each sample to the HiBind® DNA Mini Columns
  11. Spun columns at 10,000 x g for 1 minute, discarded filtrate
  12. Repeated steps 10 and 11 until all of sample was applied to columns
  13. Added 500ul HBC buffer with isopropanol, spun at 10,000 x g for 30sec, discarded filtrate
  14. Added 700ul DNA Wash Buffer with ethanol, spun at 10,000 x g for 1 minute, discarded filtrate
  15. Repeated steps 13 and 14 once
  16. Dry spun for 2 minutes at maximum speed (14,000 x g)
  17. Transferred columns to clean 1.5ul microcentrifuge tubes
  18. 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
  19. Stored DNA at 4°C

C. Illumina Library Prep

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

  1. Sheared DNA to two different sizes, ~400bp and ~600bp (6ug into each).
  2. Size selected for two ranges, 350-500bp and 500-700bp.
  3. 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)
  4. 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).
  5. 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)
  6. 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)
  7. 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

  1. Followed instructions exactly, with the exception of manually size selecting on 0.75% Agarose field inversion gel instead of selecting using AMPure beads
  2. 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. ( more info)

Discussion

, 2015/04/08 09:29

Do we have a way to view the video's shown in class?

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lecture_notes/04-03-2015.txt · Last modified: 2015/04/06 10:00 by sihussai