TITLE: DNA metabarcoding protocol for siphonophore gut contents
AUTHORS: Alejandro  Damian Serrano

[DESCRIPTION]
Protocol for the SiphWeb DNA metabarcoding of siphonophore gut content. 
Starting with DNA extractions from pooled frozen gastrozooids, this protocol takes the user through the steps of PCR amplification, PCR cleanup and pooling of amplicons from six complementary 18S barcodes that can be submitted into an Illumina MiSeq Lane.

[STEPS]
SECTION: DNA Extractionion
1. Mainly identical to:
http://www.bea.ki.se/documents/EN-DNeasy%20handbook.pdf
With the following modifications:

-Digestion at 56 °C 
-Elution using 2 rounds of incubation and centrifuge with 50 µL QIAgen DNeasy Blood & Tissue kit, cultured cells , incubating 10 min at 56 °C , to a total of 100 µL 
-Label 2 mL Eppendorf tubes to hold the extracted DNA. Include the extraction number, the siphonophore species, "GCDNA", the date of extraction, and your initials.
-Use Nanodrop to assess DNA yield (ng/µl) and 260:280 ratio.
 
Store eluted DNA in -20 °C freezer.

SECTION: PCR
2. Based off Promega GoTaq protocol.

(Plan for a 96-well final plate [90 wells used for 13 templates, a positive, and a negative control, across 6 primer pairs (detailed in T1)], can be re-scaled as needed).
See reference planning spreadsheet here: https://docs.google.com/spreadsheets/d/1x71z9YLqxo9XszNcjAPB__p0cnHHYyMTEHNL4wb84j4/edit#gid=656913706
Only select templates that have a DNA yield > 10ng/µl and a 260:280 > 1.7
SECTION: PCR
3.  
    Name   Type (F/R)   Degeneracy   % matches   Primer sequence   Length (bp)   TM © min   TM © max   %GC   Hairpin TMC   Self Dimer TMC   Pair Dimer TMC   Insert size (bp)   Start position   End position     18S152F   F   0   99   TGACGGAAGGGCACCACCAG   20   62.7   64.6   63.2   63   0.1   None   152   3275   3301     18S152R   R   0   98   TCCACCAACTAAGAACGGCC   20   60   60   55   None   None   None   152   3425   3457     18S166F   F   0   67   AACGGCTACCACATCCAAGG   20   60   60   55   None   None   None   166   887   906     18S166R   R   0   74   CACCAGACTTGCCCTCCAAT   20   60   60   55   31.9   None   None   166   1152   1129     18S272F   F   0   82+ (3 msms)   AAACGATGCCGACTAGCGAT   20   59.9   59.9   50   44.6   7.9   6.7   272   3064   3085     18S272R   R   0   98   TCCACCAACTAAGAACGGCC   20   60   60   55   None   None   6.7   272   3457   3425     18S179F   F   0   98   GGCCGTTCTTAGTTGGTGGA   20   60   60   55   None   None   11   179   3425   3457     18S179R   R   0   93 (2 msms)   TGCGGCCCAGAACATCTAAG   20   60.1   60.1   55   None   None   11   179   4139   4116     18S261F   F   0   95   AACAGGTCTGTGATGCCCTT   20   59.2   59.2   50   44.1   4.2   14   261   4081   4118     18S261R   R   0   95   TGTGTACAAAGGGCAGGGAC   20   59.9   59.9   55   None   None   14   261   4432   4397     18SV9ADSF   F   0   66   CTTTGTACACACCGCCCGTC   20   61.6   61.6   60   None   None   None   134   4,421   4443     18SV9ADSR   R   0   66   CCTTGTTACGACTTTTACTTCCTCT   25   58.8   58.8   40   None   None   None   134   4745   4779    
 
SECTION: PCR
4. -Pull all reagents in T2 from the freezer and let them thaw.

-Separate and label two PCR plates, 
    one for V9 (annealing at 48 °C for 45 s  per cycle)
    one for V3, V5-V7S, V5-V7L, V7, and V7p+V8 (all annealing at 54 °C for 1 min x30 cycles ). Divide and label each pair of rows that will contain each primer pair mix.
SECTION: PCR
5. 
 
  ReagentVolume (µm) per tube in the plateTemplate (variable)2Forward primer (10µM)0.5Reverse primer (10µM)0.5MgCl2 (25mM)2.5dNTPs (10mM)1GoTaq0.2Buffer 5X2.5BSA1H2O14.8TOTAL25
 
-Make master-mastermix for all primer pairs as in T3. Always vortex each reagent before pipetting, and vortex the mixture at the end.
 
   MgCl2 (25mM)2.5350dNTPs (10mM)1140GoTaq0.228Buffer 5X2.5350BSA1140H2O14.82072TOTAL253080Serve: 440µl/mastermix

SECTION: PCR
6. -Serve 440 µL of this master-mastermix into 6 2 mL Eppendorf tubes. Lable the tubes with the name of each primer pair.
-Add 10 µL of 10 micromolar (µM)  Forward and 10 µL of 10 micromolar (µM) Reverse primer into each primer-specific mastermix to end up with T4.
-Vortex .
 
   1010502045020296460

SECTION: PCR
7. In a set of 14 PCR tubes (can use two rows in either PCR plate too), serve an excess amount of template to seed each well with. I usually go for 20 µL . Serve the templates in the same spatial arrangement as they will be seeded in each set of rows for each primer pair. 

Include the positive and negative controls. I tend to pick the elution AE buffer stock I used for extractions as negative control, and a previously successful template as positive control.
SECTION: PCR
8. Using a single 200 µL pipette tip on a multi-step pipette set to 23 µL doses in 4 steps, seed each primer-specific pair of rows. Remember to get rid of the excess (before or after serving the steps, depending on the model of the pipette). Change tips in between primer-pair-specific mastermix.

Using a multichannel 10-100µl, seed the 2 µL of template in each well. Be careful to not accidentally aspire air at the bottom of the tubes/wells and end up with un-seeded wells. Always visually inspect the tips defore serving into the wells. Change tips every time to avoid contamination.

SECTION: PCR
9. Seal the plates (6 s under the plate press) with cellophane cover. 
Vortex plates. 
1000 rpm, 10 s and spin it down. 

SECTION: PCR
10. Insert plates (V9) and (V3, V5-V7S, V5-V7L, V7, V7p+V8) in two separate thermocyclers. Run PCRs following the programs specified in T5A and T5B respectively.
 

 


 

SECTION: PCR
11. Vortex plates. 
1000 rpm, 10 s and spin it down.
Un-seal the plates (6 s under the plate press)
SECTION: Gel Electrophoresis
12. 2 g of Agarose for every 100 mL of TAE buffer.
 

 

For 90 wells, we will need 3 gels with 2 16-well combs each.
For 3 gels, we need to make 200 mL of agar in one batch, and 100 mL in another batch.
In the double batch, add 4 g of agarose and 200 mL of TAE buffer. Microwave for 3 min. Stir and microwave for another 1 min if solids are still not dissolved. When cool, add 4 µL of SYBR-Safe.
In the single batch, add 2 g of agarose and 100 mL of TAE buffer. Microwave for 150 s. Stir and microwave for another 1 min if solids are still not dissolved. When cool, add 2 µL of SYBR-Safe.
 

Pour gels and let them cool until solid.
Annotate distribution of samples on wells. I usually arrange a row per primer pair ordered by amplicon size: V9:V5-V7S, V3:V7, V7p+V8:V5-V7L in 3 gels, and then the well sequence would go: Ladder, A1-B6 (letters vary with position in the PCR plate), -ve, +ve.

SECTION: Gel Electrophoresis
13. Set up power source to 125V, 500mA, 45 min
Load 6 µL of 100bp ladder on the left side of each row of wells (2 per gel).
Use adjustable-spacer multichannel pipette to load 5 µL of PCR products onto the wells. 
Well distance is   , PCR plate distance is   .
Run gels.


SECTION: Gel Electrophoresis
14. Place gels under UV light, take photograph, print.

 

SECTION: PCR Cleanup
15. Put some nuclease-free water to warm up to 56 °C .
 
Add 55 µL of water into each PCR well (except controls and failed reactions) using a multichannel 20-200µl and filter tips. Mix by pipetting up and down ~5 times. Transfer mix to ExcelaPure purification plate. Change tips every time.


SECTION: PCR Cleanup
16. 5000 rpm, 7 min  Centrifuge the purification plate with a collecting plate underneath.
Make sure to balance the plate centrifuge with a couple of adequately weighted collecting plates.
SECTION: PCR Cleanup
17. Add 50 µL of warm (56 °C ) water to each well in the purification plate. Mix by pipetting up and down ~15 times with the multichannel pipette and filter tips. Change tips every time.
SECTION: PCR Cleanup
18. Incubate at 56 °C for 10 min .
Prepare a final clean product PCR plate: Label and annotate adequately to preserve sample location. I recommend drawing a line between each pair of rows, segregating primer pairs for barcodes V3 to V9.
SECTION: PCR Cleanup
19. Mix the warm eluted clean products in the purification plates by pipetting up and down ~15 times with the multichannel pipette and filter tips. 
Transfer clean products to the final plate.
Change tips every time.
SECTION: PCR Cleanup
20. Seal the plates (6 s under the hot plate press) with cellophane cover. Store in -20 °C freezer.

SECTION: Amplicon isomolar poolingling
21. The goal here is to obtain an equal concentration of each barcode for each sample, so they can be pooled with equal representation for MiSeq Illumina sequencing.

First step is to quantify the DNA content in each cleaned PCR product well using Qubit HS.
 
If you have the pre-mixed mastermix, just run the standards at 190 µL of mastermix and 10 µL of standard.
Vortex mix and incubate for 1min.
Prepare the assay tubes with  198 µL MM and 2 µLof template.
Vortex mix and incubate for 1min.
Pick the ng/µl units.
SECTION: Amplicon isomolar poolingling
22. -Calculate amount of template to seed in each pooling well. 
-Pool target concentrations are equal to the lowest concentration across barcodes for a sample excluding those that are <2ng/µl, which is the minimum required by YCGA for amplicon sequencing.
      0.50.50.60.60.91
 
Template volume to add = (50 µL /(Qubit ng/µl value of original PCR product/Pool target concentration))*Molar_correction_factor      
~  See T6 for Molar correction factors ~
SECTION: Amplicon isomolar poolingling
23. -Calculate amount of water to add in each pooling well. 
Water volume to add = (50 µL *Molar_correction_factor) - Template volume to add
SECTION: Amplicon isomolar poolingling
24. Prepare a pooling PCR plate, labeled.
Add the water and template quantities for each well. Change the pipette tip every time when handling PCR products.
SECTION: Amplicon isomolar poolingling
25. Label Eppendorf 2 mL tubes to hold to final amplicon pools. Include the extraction number, the genus of the siphonophore, "GC DNA pooled", and your initials.
Using a multichannel pipette of 20-200µl, proceed to pool the amplicons by sample. If the volumes do not fit in a row on the pooling PCR plate, you can make 2 semi-pools, one combining V9:V5-V7S:V3, and one combining V7:V7p+V8:V5-V7L.

 

Using a single-channel 20-200µl pipette, transfer the pooled amplicons to the Eppendorf tubes. Vortex and close caps.
Store the amplicon pools in a -20 °C freezer.