DNA extraction and 16S amplicon sequencing

DNA EXTRACTION FROM SWAB 
 

Materials & Reagents:

• Lysostaphin (Cat: L7386-15MG) - store at -20°C
• TE Buffer (Cat: AM9858)
• Lysozyme (Cat: 10 837 059 001)
• Sodium Chloride (Cat:S3014-500G) – diluted to 5 M
• Proteinase K (REC.), PCR Grade (20mg/mL) (Cat: SKU EO0492)(
• Sodium Dodecyl Sulfate (Cat:L3771-100G) – diluted to 10%
• Phenol:chloroform:isoamyl alcohol (25:24:1 pH8.0) (Cat: 77617-500mL) - store at 4°C
• Ammonium acetate (Cat: A1542-500G) – diluted to 10 M
• Molecular Grade Ethanol (Cat: AJA214-20LPL)
• EZ-10 Spin Column Genomic DNA Kit, Bacterial, Wash Solution (Cat:BS624-250)
• UltraPure DNase/RNase-Free Distilled Water (Cat: 10977023)

Equipment:

• Sterile forceps
• Sterile scissors
• Ratek Vortex (Model: VM1)
• Fastprep-24 tissue lysis Bead Beater (Cat: SKU: 116004500)
• Screw cap conical 2 mL microcentrifuge tubes (Cat: QSP522-S)
• 2 mL Microcentrifuge tubes (Cat: QSP508-GRD)
• 1.5 mL RNase-free Microfuge Tubes (Cat: LTSAM12400)
• Ratek Dry Heat block (Model: DBH30DP)
• Syringe hypodermic luer slip 3mL (Cat: TER6SS.03S)
• Parafilm Sealing Film (Cat: PM996TSPK)
• 50 ML Conical Centrifuge Tube, sterile (Cat: NUN339652)
• Centrifuge (Model: Heraeus Multifuge X3 Centrifuge)
• Microcentrifuge (Model: Eppendorf Centrifuge 5424)
• Zirconia/Silica Beads, 0.1mm diameter (Cat: BP/1107910)
• Zirconia/Silica Beads, 1.0mm diameter (Cat: BP/1107911)
• EZ-10 Spin Column Genomic DNA Kit, Bacterial, Spin Columns (Cat:BS624-250)

Pre-Process:

• Using sterile forceps transfer dry swab to a 2 mL screw capped tube
• Using sterile scissors cut the shaft of the swab, so the lid can be screwed on
• Add 400 µL of TE buffer to the tube. Vortex for 30 seconds
• Rest for 10 minutes – can be stored at -80°C until extraction
   

Pre-extraction:

• Weigh lysostaphin
  o  X = n*0.056 mg of lysostaphin
• Set 1 heat block to 95°C and 1 to 37°C
• Get ice

Extraction Method:

1. Remove TE by centrifugation
   • Take plunger out of a sterile 3 mL syringe
   • Using sterile forceps, transfer swab to syringe barrel
   • Paraffin wrap the bottom of syringe to the top of the 2 mL screw capped tube
   • Put syringe with swab + tube in a 50mL eppendorf tube and screw on 50 mL cap
   • Centrifuge 50mL tube in large centrifuge at max for 5 minutes
   • Dispose of syringe + swab and keep 2 mL tube (should now contain ~400 µL)
2. Incubate tube at 95°C for 1 minute
   • Put on ice for 1 minute to cool
   • Set temp to 55°C
3. Add n*5.6 µL of PBS to lysostaphin tube
4. Add n*23.2 µL of lysozyme to lysostaphin tube and mix
5. Add 28.8 µL of lysostaphin + lysozyme to sample tubes
6. Add 10 µL of 5 M NaCl
7. Incubate at 37°C for 1 h
8. Add 20 µL of Proteinase K (20 mg/mL)
9. Add 50 µL of 10% SDS
10. Add 90 µL of  5M NaCl
11. Incubate at 55°C for 30minutes
12. Add one small scoop of ceramic beads (small and medium beads)
13. Put in Fast-Prep-24 at 6.5 m/s for 60 seconds
14. Centrifuge at 8000 x g for 1 minute to reduce bubbles
15. Add 700 µL of phenol:chloroform:isoamyl alcohol (25:24:1 pH8.0)
16. Vortex/ shake vigorously for 1 minute
17. Centrifuge at 13, 000 x g for 20 minutes
18. Transfer top aqueous layer (400-500 µL) to a clean 2 mL tube
19. Add 1:10 vol (40-50 µL) of 10 M ammonium acetate
20. Add 1:1 vol (450-600 µL) of 100% cold ethanol
21. Let sit in fridge for at least 5 min – can be stored at 4°C overnight

DNA Clean Up:

1. Add DNA (~650 µl) solution to spin column.
2. Centrifuge at 8,000 x g for 2 minutes
3. Discard elute
4. Repeat steps 22-24 until all DNA has been added to spin column
5. Add 500 µL of Wash Solution.
6. Centrifuge at 8, 000 x g for 2 minutes
7. Discard elute
8. Centrifuge again at 8,000 x g for 1 minute
9. Place column in a clean 1.5 mL collection tube
10. Add 50 µL of dH2O heated to 55^oC
11. Incubate at room temp for 10 minutes
12. Centrifuge at 8, 000 x g for 2 minutes
13. Repeat steps 31-33 for 2^nd elution
14. Store sample at -80°C

16S AMPLICON SEQUENCING

(Adapted from Illumina 16S Metagenomic Sequencing Library Preparation)

Materials & Reagents:

• 2X kappa HiFi HotStart Ready Mix (Cat: ROC-07958935001) – store at -20°C
• Amplicon PCR Reverse Primer (10 µM) – store at -20°C
• Amplicon PCR Forward Primer (10 µM) - store at -20°C
• Nextera XT Index Kit v2 (Set A, B, C and D) (Cat: FC-131-2001, FC-131-2002, FC-131-2003, FC-131-2004) - store at -20°C
• AMPURE XP Beads (Cat: A63881) - store at  4°C
• Molecular Grade Ethanol (Cat: AJA214-20LPL) – diluted to 80%
• Tris (1 M), pH 8.0 (Cat: AM9856) – diluted to 10 mM

Equipment:

• Veriti 96-Well Thermal cycler (Model: 9902)
• Microplate centrifuge (Model: CM-6MT)
• Ratek Vortex (Model: VM1)
• Microseal adhesive films (Cat: 0030127.781)
• 96-well plate, conical bottom, half skirt, flat top (Cat: LC3973-520-000)
• 96-well hard shell plate (Cat: HSP9601)
• TruSeq index plate fixture

Amplicon PCR:

    1. Set up the following reaction of DNA, 2x KAPA HiFi HotStart Ready-Mix, and Primers:

   2. Seal the plate and set up the following reaction on a thermocycler:

• 95°C for 3 minutes
• 25 cycles of:
   95°C for 30 seconds
   55°C for 30 seconds
   72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
   

PCR Clean-Up:

1. Using a multichannel pipette add 20 µl of beads to each well 
2. Gently pipette up and down 10 times. Change tips between each sample
3. Incubate at room temperature for 5 minutes
4. Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared
5. With the plate on the magnetic stand, use a multichannel pipette to carefully remove and discard the supernatant
6. With the plate on the magnetic stand, wash the beads with freshly made 80% ethanol as follows:
   • Using a multichannel pipette, add 200 µl of 80% ethanol to each well.
   • Incubate the plate at room temperature for 30 seconds.
   • Carefully remove and discard the supernatant.
7. Perform a second ethanol wash following the step 8 instructions
8. Using a P20 multichannel pipette remove excess ethanol
9. With the plate still on the stand, allow the beads to air-dry for 10 minutes
10. Remove the plate from the stand. Using a multichannel pipette, add 52.5 µl of 10 mM Tris to each well
11. Gently pipette mix up and down 10 times, changing tips after each sample
12. Incubate at room temperature for 2 minutes
13. Place the plate on the stand for 2 minutes, or until the supernatant has cleared
14. Using a multichannel pipette, carefully transfer 48 µl of the supernatant to a new plate. Change tips between samples
15. Measure concentration

Index PCR:

1. Add 5 µl or 10 µl of dH20 to appropriate well (volume is dependent on amplicon concentration, refer to spreadsheet for volume)
2. Add 25 µl of 2X kappa HiFi HotStart Ready Mix to each well
3. Arrange index primers in the index plate fixture, with index 1 (i7) primers in order horizontally, and index 2 (i5) primers in order vertically
4. Using a multichannel pipette, add 5 µl on index 2 primers to each column of the plate. Change tips between samples
5. Using a multichannel pipette, add 5 µl of index 1 primers to each row on the plate. Change tips between samples
6. Using a multichannel pipette, add 5 µl or 10 µl of amplicon DNA to appropriate well (volume is dependent on amplicon concentration, refer to spreadsheet for volume) 
7. Cover plate with film, gently vortex and centrifuge at 280 xg for 1 minute
8.  Place the plate in a thermocycler and run the following program:
   • 95°C for 3 minutes
   • 8 cycles of:
       95°C for 30 seconds
       55°C for 30 seconds
       72°C for 30 seconds
   • 72°C for 5 minutes
   • Hold at 4°C
    

PCR Clean-up 2:

1. Repeat steps 3-20 with the following changes:
   • Add 56 µl of beads to each well (instead of 20 µl in step 3)
   • Add 27.5 µl of 10 mM Tris to each well (instead of 52.5 µl in step 15)
   • Transfer 23 µl of the supernatant to a new plate (instead 48 µl in step 19)

Library Normalization and Pooling:

1. Using a new plate and 10 mM Tris, dilute samples with to 8 ng/µl (for samples below 8 ng/µl and above 4 ng/µl dilute to 4 ng/µl)
2. Pool 3 µl of each sample into a 1.5 mL tube (for each plate pool samples evenly between 3 tubes)
3. Run bioanalyzer on pooled samples
4. Measure concentration of pooled samples
5. Dilute 8 ng/µl pooled tubes to 4 ng/µl
6. Measure concentration of pooled samples
7. Pool all tubes into 1 final 1.5 mL pooled tube
8. Add samples below 4 ng/µl to final pooled tube, using the following formula to determine volume: 
   • Index DNA concentration/1.9
9. Measure concentration
10. Using 10 mM Tris, adjust final concentration to 1.9 ng/ µl
11. Store plate at store at -20°C