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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Protocol for the SiphWeb DNA metabarcoding of siphonophore gut content. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">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.</div></div></div>

[STEPS]
?. [DNA Extractionion]
Mainly identical to:http://www.bea.ki.se/documents/EN-DNeasy%20handbook.pdfWith the following modifications:-Digestion at -Elution using 2 rounds of incubation adn centrifuge with  , incubating  at  , to a total of -Label  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  freezer.
[1-2h]
[of AE buffer]
56 °C
100 µl
2 mL
-20 °C
?. [PCR]
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=656913706Only select templates that have a DNA yield > 10ng/µl and a 260:280 > 1.7
?. [PCR]
-Pull all reagents in T2 from the freezer and let them thaw.-Separate and label two PCR plates,     one for 134 (annealing at  , elongation  )    one for 152, 166, 179, 261, and 272 (all annealing at  , elongation  ). Divide and label each pair of rows that will contain each primer pair mix.
48 °C
54 °C
?. [PCR]
AB1ReagentVolume (µm)2Template (variable)23Forward primer (10µM)0.54Reverse primer (10µM)0.55MgCl2 (25mM)2.56dNTPs (10mM)17GoTaq0.28Buffer 5X (green)2.59BSA110H2O14.811TOTAL25-Make master-mastermix for all primer pairs as in T3. Always vortex each reagent before pipetting, and vortex the mixture at the end.  AB1ReagentVolume (µm)2MgCl2 (25mM)3503dNTPs (10mM)1404GoTaq285Buffer 5X3506BSA1407H2O20728TOTAL3080
AB1ReagentVolume (µm)2Template (variable)23Forward primer (10µM)0.54Reverse primer (10µM)0.55MgCl2 (25mM)2.56dNTPs (10mM)17GoTaq0.28Buffer 5X (green)2.59BSA110H2O14.811TOTAL25
AB1ReagentVolume (µm)2MgCl2 (25mM)3503dNTPs (10mM)1404GoTaq285Buffer 5X3506BSA1407H2O20728TOTAL3080
?. [PCR]
-Serve  of this master-mastermix into 6  Eppendorf tubes. Lable the tubes with the name of each primer pair.-Add  of   Forward and  of  Reverse primer into each primer-specific mastermix to end up with T4.-Vortex .  AB1ReagentVolume (µl)2Forward primer (10µM)103Reverse primer (10µM)104MgCl2 (25mM)505dNTPs (10mM)206GoTaq47Buffer 5X508BSA209H2O29610TOTAL460
440 µl
2 mL
10 µl
10 µl
AB1ReagentVolume (µl)2Forward primer (10µM)103Reverse primer (10µM)104MgCl2 (25mM)505dNTPs (10mM)206GoTaq47Buffer 5X508BSA209H2O29610TOTAL460
?. [PCR]
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  . 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.
20 µl
?. [PCR]
Using a single  pipette tip on a multi-step pipette set to  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  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.
200 µl
23 µl
2 µl
?. [Gel Electrophoresis]
of Agarose for every  of TAE buffer.For 90 wells, we will need 3 gels with 2 16-well combs each.For 3 gels, we need to make  of agar in one batch, and  in another batch.In the double batch, add  of agarose and  of TAE buffer. Microwave for . Stir and microwave for another  if solids are still not dissolved. When cool, add  of SYBR-Safe.In the single batch, add  of agarose and  of TAE buffer. Microwave for . Stir and microwave for another  if solids are still not dissolved. When cool, add  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: 134+152, 166+179, 261+272 in 3 gels, and then the well sequence would go: Ladder, A1-B6 (letters vary with position in the PCR plate), -ve, +ve.
2 g
100 mL
200 mL
100 mL
4 g
200 mL
4 µl
2 g
100 mL
2 µl
?. [PCR Cleanup]
Put some nuclease-free water to warm up to  .Add  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.
56 °C
55 µl
?. [Amplicon isomolar poolingling]
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  of mastermix and  of standard.Vortex mix and incubate for 1min.Prepare the assay tubes with   MM and of template.Vortex mix and incubate for 1min.Pick the ng/µl units.
190 µl
10 µl
198 µl
2 µl
?. [PCR]
Seal the plates ( under the plate press) with cellophane cover. Vortex plates.  and spin it down.
Centrifuge: 1000 33, 10 s
?. [PCR]
Insert 134 and 152+166+179+261+272 plates in separate thermocyclers. Run PCRs following the programs specified in T5A and T5B respectively.  ABCDEF1Temperature (C)94944872722Time2m30s30s1m5m3Cyclesx30x30x30  ABCDEF1Temperature (C)94945472722Time2m30s1m1m5m3Cyclesx30x30x30
ABCDEF1Temperature (C)94944872722Time2m30s30s1m5m3Cyclesx30x30x30
ABCDEF1Temperature (C)94945472722Time2m30s1m1m5m3Cyclesx30x30x30
?. [PCR]
Vortex plates.  and spin it down.Un-seal the plates ( under the plate press)
Centrifuge: 1000 33, 10 s
?. [Gel Electrophoresis]
Set up power source to 125V, 500mA, Load  of 100bp ladder on the left side of each row of wells (2 per gel).Use adjustable-spacer multichannel pipette to load  of PCR products onto the wells. Well distance is  , PCR plate distance is  .Run gels.
6 µl
5 µl
?. [Gel Electrophoresis]
Place gels under UV light, take photograph, print.
You should see a band over each well except the negative control. The bands should land at approximately the height (in bp) of the ladder corresponding to the name (expected amplicon length) of each primer pair.
?. [PCR Cleanup]
Centrifuge the purification plate with a collecting plate underneath.Make sure to balance the plate centrifuge with a couple of adequately weighted collecting plates.
Centrifuge: 5000 33, 7 min
?. [PCR Cleanup]
Add  of warm ( ) 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.
50 µl
56 °C
?. [PCR Cleanup]
Incubate at  for  .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 134 to 272.
56 °C
?. [PCR Cleanup]
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.
?. [PCR Cleanup]
Seal the plates ( under the hot plate press) with cellophane cover. Store in  freezer.
-20 °C
?. [Amplicon isomolar poolingling]
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.
?. [Amplicon isomolar poolingling]
-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   ABCDEF113415216617926127120.50.50.60.60.91Template volume to add = ( /(Qubit ng/µl value of original PCR product/Pool target concentration))*Molar_correction_factor      ~  See T6 for Molar correction factors ~
ABCDEF113415216617926127120.50.50.60.60.91
50 µl
?. [Amplicon isomolar poolingling]
-Calculate amount of water to add in each pooling well. Water volume to add = ( *Molar_correction_factor) - Template volume to add
50 µl
?. [Amplicon isomolar poolingling]
Label Eppendorf  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 134+152+166, and one combining 179+261+272.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  freezer.
2 mL
Be very careful on this step, since mistakes during pooling will result in having to re-do the whole protocol for the samples affected.
-20 °C
?. [PCR]
ABCDEFGHIJKLM1Name
Type (F/R)
Primer sequence
Length (bp)
TM © min
TM © max
%GC
Hairpin TMC
Self Dimer TMC
Pair Dimer TMC
Insert size (bp)
Start position
End position
2152FF
TGACGGAAGGGCACCACCAG
20
62.7
64.6
63.2
63
0.1
None
152
3275
3301
3152RR
TCCACCAACTAAGAACGGCC
20
60
60
55
None
None
None
152
3425
3457
4166FF
AACGGCTACCACATCCAAGG
20
60
60
55
None
None
None
166
887
906
5166RR
CACCAGACTTGCCCTCCAAT
20
60
60
55
31.9
None
None
166
1152
1129
6272FF
AAACGATGCCGACTAGCGAT
20
59.9
59.9
50
44.6
7.9
6.7
272
3064
3085
7272RR
TCCACCAACTAAGAACGGCC
20
60
60
55
None
None
6.7
272
3457
3425
8179FF
GGCCGTTCTTAGTTGGTGGA
20
60
60
55
None
None
11
179
3425
3457
9179RR
TGCGGCCCAGAACATCTAAG
20
60.1
60.1
55
None
None
11
179
4139
4116
10261FF
AACAGGTCTGTGATGCCCTT
20
59.2
59.2
50
44.1
4.2
14
261
4081
4118
11261RR
TGTGTACAAAGGGCAGGGAC
20
59.9
59.9
55
None
None
14
261
4432
4397
12134FF
CTTTGTACACACCGCCCGTC
20
61.6
61.6
60
None
None
None
134
4,421
4443
13134RR
CCTTGTTACGACTTTTACTTCCTCT
25
58.8
58.8
40
None
None
None
134
4745
4779
ABCDEFGHIJKLM1Name
Type (F/R)
Primer sequence
Length (bp)
TM © min
TM © max
%GC
Hairpin TMC
Self Dimer TMC
Pair Dimer TMC
Insert size (bp)
Start position
End position
2152FF
TGACGGAAGGGCACCACCAG
20
62.7
64.6
63.2
63
0.1
None
152
3275
3301
3152RR
TCCACCAACTAAGAACGGCC
20
60
60
55
None
None
None
152
3425
3457
4166FF
AACGGCTACCACATCCAAGG
20
60
60
55
None
None
None
166
887
906
5166RR
CACCAGACTTGCCCTCCAAT
20
60
60
55
31.9
None
None
166
1152
1129
6272FF
AAACGATGCCGACTAGCGAT
20
59.9
59.9
50
44.6
7.9
6.7
272
3064
3085
7272RR
TCCACCAACTAAGAACGGCC
20
60
60
55
None
None
6.7
272
3457
3425
8179FF
GGCCGTTCTTAGTTGGTGGA
20
60
60
55
None
None
11
179
3425
3457
9179RR
TGCGGCCCAGAACATCTAAG
20
60.1
60.1
55
None
None
11
179
4139
4116
10261FF
AACAGGTCTGTGATGCCCTT
20
59.2
59.2
50
44.1
4.2
14
261
4081
4118
11261RR
TGTGTACAAAGGGCAGGGAC
20
59.9
59.9
55
None
None
14
261
4432
4397
12134FF
CTTTGTACACACCGCCCGTC
20
61.6
61.6
60
None
None
None
134
4,421
4443
13134RR
CCTTGTTACGACTTTTACTTCCTCT
25
58.8
58.8
40
None
None
None
134
4745
4779