TITLE: DNA Barcoding Standard Operating Protocol, Plants and Lichens at RBGE, Lab methods: PCR and Sequencing
AUTHORS: Laura L Forrest, Michelle Hart

[DESCRIPTION]
This is part of the collection DToL Taxon-specific Standard Operating Procedures for the Plant Working Group (protocols.io). The SOP collection contains guidance on how to process the various land plant taxa within the scope of the Darwin Tree of Life project (DToL). The guidance specifically refers to the laboratory processing of extracted DNA from Darwin Tree of Life plant and lichen collections that takes place at the Royal Botanic Garden (RBGE). Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger institute.

This DNA barcoding SOP outlines PCR amplification and Sanger sequencing of DNA extractions from plant and lichen samples for the Darwin Tree of Life project at the Royal Botanic Garden Edinburgh. 

DNA barcoding is used as part of the species identification process AND sample tracking (to check that the genome sequence corresponds to the material that was sent and that there have been no sample mix-ups).

Definition: Land plants (Embryophyta) and lichens
Including: Bryophyta, Marchantiophyta, Anthocerotophyta, Lycopodiophyta, Polypodiophyta, Pinophyta, Cycadophyta, Ginkgophyta, Gnetophyta, Magnoliophyta, lichenized fungi

Excluding: non-lichenized fungi



[GUIDELINES]
Including: Bryophyta, Marchantiophyta, Anthocerotophyta, Lycopodiophyta, Polypodiophyta, Pinophyta, Cycadophyta, Ginkgophyta, Gnetophyta, Magnoliophyta, lichenized fungi


 

Marker choice 
For plant accessions that are required for DToL identification/sample tracking purposes: 

Bryophytes (liverworts, hornworts and mosses) are amplified for plastid markers rbcL and psbA-trnH and nuclear marker ITS2.
Ferns and lycophytes are amplified for plastid marker rbcL.
Seed plants are amplified for plastid marker rbcL and nuclear marker ITS2. 
Lichens are amplified for nuclear marker ITS.
The plastid marker trnL-trnF is a subsidiary marker that can be used for all land plants.


PCR amplification. 
Routinely use PCR enhancers TBT-PAR (Samarakoon et al., 2013) or CES (Ralser et al., 2006) for consistent amplification. 
Set up PCRs in strips rather than plates, due to a high rate of failure of samples around the margins of plates (probably through poor lid seals).
For plants, per reaction: 1x PCR buffer, 0.2 mM each dNTPs, 1.5 mM MgCl2, 1x additive, 1 μM of each primer, 2.5 U taq polymerase and c. 1 ng template DNA.
For lichens, per reaction:  1x PCR buffer, 0.2 mM each dNTPs, 2.4 mM MgCl2, 1x additive, 0.4 M of each primer, 0.05 U/μl taq polymerase and c. 2-5 ng template DNA


PCR visualization
We save our gel images in a DToL Gel folder on the RBGE DNA server, then copy them into electronic lab books.


PCR failure (Before sending for sequencing):
If there is no band on the gel for a given amplification, the PCR should be repeated. 

If PCR of an extraction fails across both/all loci, DNA may be quantified on an agarose gel and by fluorometry or just discarded, and a repeat DNA extraction made from the silica-gel dried tissue sample. 

If PCR just fails for one or two loci, it may be repeated using an alternative additive (TBT-PAR or CES) as well as with a 1:25 dilution of the original DNA.

If there are multiple bands, particularly for variable-length markers (usually ITS2 and psbA-trnH), PCR may be repeated with alternative primer pairs (where available), or at a higher annealing temperature. If this happens repeatedly then the DNA can be re-extracted from the silica-gel dried tissue, to rule out sample contamination, or the sample may pass barcoding with just a single DNA barcoding locus.

At least one repeat PCR should be carried out for all poor / failed amplifications in a batch.

If over 20% of samples have failed for a marker that is usually successful (e.g. rbcL), the entire batch of PCRs should be discarded and the PCR repeated, to save time cherry-picking successful samples. 


Sequencing failure (after sending for sequencing):
If a sample that was sent for sequencing has generated a low quality read, or failed to sequence:
First check the reads to rule out microsatellites close to the primers causing the failure,  
If that is not the reason, check the PCR gel images to estimate the quality of the PCR product (band tightness and brightness). 

If the PCR band quality in the jpg is high and microsatellites were not the problem, sequencing can be reattempted from the stored, cleaned PCR product. 
If the PCR band quality in the jpg is low, PCR can be repeated as above (PCR failure section). 
If microsatellites are causing the problem, unidirectional sequence can be accepted if it is of high quality, or alternative primers can be tried if there are any available. 
Unreadable sequences for ITS2 can also be due to co-amplifying endogenous fungi, particularly in liverworts, so failures in these cases are to be expected and will not be rectified by redoing the extraction or the PCR; occasionally PCR with alternative primers rectifies this, but time constraints mean that this is usually not feasible.
If sequence reads contain more than one peak at any position (except for where wobbles are likely to be real i.e. in diploid or concatenated markers, in this case the nuclear ribosomal repeat region ITS), indicating contamination, PCR can be repeated from the extracted DNA if only a single marker is affected (therefore the double peaks are likely due to contamination during PCR or sequencing), or the extraction should be repeated if all markers are similarly affected (therefore the double peaks are likely due to contamination at the DNA extraction stage).

 






 
     Version      Date      Major changes      Contributors        1.0      2020-04-07      Draft   version      Lyndall   Pereira (NHM)        2.0      2020-04-29      DNA   barcoding for reference library at NHM edited      NHM: Olga   Sivell, Laura Sivess, Gavin Broad, Ben Price, Lyndall Pereira        3.0      2020-05-13      Addition of   pictorial dissection for each taxon group      Liam   Crowley (Oxford WW) and Lyndall Pereira (NHM)        3.1      2020-07-17       SOP text   split into Order-specific SOPs. Uniform style, publication quality images   with colour blind friendly dissection lines      Olga   Sivell, Laura Sivess        4.0      2022-01-04      Overall   formatting further improved (including spelling/ punctuation changes, table   of contents links), photo guides made more uniform, edits in protocol made to   reflect any adaptations in the sampling process since July 2020, additional   taxon protocols included, unrequired taxon guides removed. First submission   to protocols.io.      Inez   Januszczak (NHM), Liam Crowley (Oxford WW), Olga Sivell (NHM), Laura Sivess   (NHM), Chris Fletcher (NHM)   



 Working SOP, checked by experts
 

[STEPS]
SECTION: PCR mix
2. Create a Master Mix for each locus. Per reaction, add:

FOR LAND PLANTS (20 μl total volume reactions):

2 µL PCR buffer (at 10x), 2 µL dNTPs (each at 2 mM), 0.6 µL MgCl2 (at 50 mM), 4 µL TBT-PAR or CES additive (both at 5x), 2 µL forward primer,2 µL reverse primer (each at 10 μM), 0.25 µL BIOTAQ (at 5 units/μl) and 6.5 µL Sigma PCR-grade water


FOR LICHENS (12.5 μl total volume reactions):

1.25 µL PCR buffer (at 10x), 1.25 µL dNTPs (each at 2 mM), 0.6 µL MgCl2 (at 50 mM), 2.5 µL TBT-PAR (at 5x), 0.5 µL forward primer,0.5 µL reverse primer (each at 10 μM), 0.13 µL BIOTAQ (at 5 units/μl) and 4.77 µL Sigma PCR-grade water


Multiply quantities by [no. of DNA extractions to be amplified plus 1.5-3 extra (to allow for pipetting errors and a negative control)]. 

Vortex briefly to mix then centrifuge briefly to get the contents to the bottom of the tube.
 

SECTION: Polymerase Chain Reactions - PCR lab
4. Take the strips through to a PCR lab and put into a thermocycler. 

Standard PCR parameters (plants):
rbcL: 2 min initial denature at 94°C followed by 35-40 cycles with a 1 min denature at 94°C, 1 min anneal at 51°C and 90 sec extension at 72°C, with a final extension of 72°C for 5 min. 
psbA-trnH: 3 min initial denature at 94°C followed by 2 cycles with a 45 sec denature at 94°C, 45 sec annealing at 50°C and 1 min extension at 72°C, then 30 cycles with a 45 sec denature at 94°C, 45 sec anneal at 45°C and 1 min extension at 72°C, with a final extension of 72°C for 5 min.
ITS2: 4 min initial denature at 95°C followed by 30 cycles with a 1 min denature at 94°C, 1 min annealing at 55°C and 45 sec extension at 72°C, with a final extension of 72°C for 5 min.

Standard PCR parameters (lichens):
ITS: 4 min initial denature at 94°C followed by 30 cycles with a 45 sec denature at 94°C, 1 min 30 sec anneal at 55°C and 90 sec extension at 72°C, with a final extension of 72°C for 10 min. 

Make a note of the thermocycler and block, and of the name of the PCR programme.
SECTION: Polymerase Chain Reactions - PCR lab
5. After the cycle is complete, put the strips into a fridge in a post-PCR lab until you are ready to run them on a gel.
SECTION: PCR mix
3. Using a repeater pipette:

LAND PLANTS: Aliquot 19 μl of master mix into the bottom of each well in the strips. 
LICHENS: Aliquot 11.5 μl of master mix into the bottom of each well in the strips.

Pipette 1 μl of the DNA extraction onto the upper side of each well (avoiding touching any master mix if you are using the same tip to add the DNA into PCRs for multiple loci); do not add DNA to the negative control.

Put lids on the strips, and briefly centrifuge to spin the DNA down into the master mix.
SECTION: Before starting - pre-PCR lab
1. Set up a laminar flow hood in a pre-PCR lab with tips, strips, sharpie etc. Get a freezer rack out the freezer.
Take PCR reagents out of the freezer and allow to defrost. Keep the polymerase in the freezer rack. Once reagents have defrosted, put them in the freezer rack to keep cool. Take water aliquot out of the fridge and put it on the freezer rack
Take the DNA out of the fridge, and sort the tubes for amplification into rows of 8 in a new rack; leave empty rows between the rows of tubes, to allow for easier handling of the tubes when setting up the PCR.
Label (strip number, locus, date) sufficient strips to have a well for each DNA sample to be amplified plus an extra well for the negative control.
SECTION: Gel visualization - post-PCR lab
6. Make up 100 ml of 1% agarose 1x TBE-buffer gel (1 g agarose in 100 ml buffer) by mixing together in a conical flask and microwaving until the agarose is completely melted, then adding 5 μl SYBR Safe gel stain. Leave to cool for c. 20 mins or cool under running water.
Slot in gel ends and pour the gel into the 96-well gel rig, add comb, and leave to set for at least half an hour.
Add approx. 3.5 μl of each PCR product to 1.5 μl of a glycerol/bromophenol blue loading dye in a plastic plate, using an electric Rainin multichannel.  
Remove the gel comb and ends, and cover with 1x TBE running buffer.
Load the samples using a Rainin manual multichannel pipette. Also add 3.5 μl of 1 kb ladder every third row.
Run at 80 volts for 40 mins to an hour. 
Turn off and disconnect the power box.
Tip off excess buffer, carry the gel to the light box, and visualise under blue light, following the instructions for the gel imaging system.
The gel image jpg should be labelled with the locus name and date, exported and saved.
Either dispose of the gel or store for reuse. Empty the gel tank and rinse all the parts with tap water.
Short term, store the PCR products at 4°C in a post-PCR lab. If it is likely to take more than a couple of days to get around to cleaning and processing them, they can be stored in a -20°C freezer, to reduce the chance of the reactions drying out. If the PCRs have failed, immediately dispose of the strips in a plastic recycling bin.

 

SECTION: PCR clean-up - post-PCR lab
7. Sample dilution: Depending on the brightness of the PCR band on an agarose gel when compared to a standard, e.g. 3.5 µl of NEB’s 1kb DNA ladder – successful barcode PCRs following the above protocols usually have VERY bright bands. For a good PCR product, dilute with 30 µl of Sigma water per well (using an electronic repeat 1000 µl multichannel). It may be necessary to add 45 µl water to extremely bright products, conversely only 10-15µl to weaker products, and no dilution for very faint bands. 
  
 


SECTION: Sanger sequencing reaction - post-PCR lab
9. Sequencing PCR (in a total volume of 10 μl): 

Label a PCR plate, or strips if you have fewer than c. 72 samples.
Make up one tube of master mix for each primer. Per sample, that is: 1.5 µL BigDye® Terminator sequencing buffer, 0.125 µL BigDye®, 0.32 µL primer (at 10 μM), 0.875 µL BDX-64 cycle sequencing additive, made up to 8 µl with 5.18 µLSigma water. 
Distribute 8 µl master mix, using a repeater pipette, to the bottoms of the wells. Do not leave gaps in the plate or the strips unless you will be able to infill them later.
Add the cleaned PCR product near to the top of wells, using a repeater multichannel pipette set to deliver two loads of 2 ul (so you can use the same pipette tip for the forward and reverse reactions). 
Seal the plate with a foil lid labelled in a standard format (e.g. RBGE_dateletter, i.e. RBGE_210628A, etc, and with a note to say “sample for clean-up and sequencing, lane names in attachment”), or put lids on strips.
Spin down strips or plates, and transfer to one of the BioRad PCR machines in the PCR lab for cycling using the BDXsq programme:

Sequencing thermocycling parameters, following BDX-64 manufacturer’s instructions: a 3 min initial denature at 96°C then 30 cycles with 10 sec denature at 96°C, 5 sec annealing at 50°C and 2 min extension at 60°C.
 

After the sequencing reaction is complete, store the plates/strips in the post-PCR lab at 4°C until they can be submitted for a sequencing run.

 






SECTION: PCR clean-up - post-PCR lab
8. ExoSAP-IT: After the samples are diluted, clean them using the commercial ExoSap-IT (Affymetrix) mix.  

Set up your tips etc. in a laminar flow hood in a post-PCR lab. Get a cool rack from the freezer, and put your ExoSap-IT in it to keep it cold.
Add 2 µl of ExoSap-IT to each PCR product using a 10 µl repeater multichannel pipette, putting the ExoSap-IT near the top of each well to reduce the risk of carrying over contamination. 
Reseal lids. 
Spin down strips.
Run the incubation in a thermocycler. PCR clean-up incubation parameters: 37°C for 30 min for optimal enzymatic activity, followed by 80°C for 15 min to deactivate the enzymes, then storage at 8-12°C.
Cleaned PCR product is stored short-term in labelled racks (project / date/ locus/ cleaned) in the post-PCR fridge. The strips are subsequently discarded, following successful sequencing, into the plastic recycling bin.