TITLE: Synthetic COI spike-ins for use in metabarcoding-based insect biodiversity surveys 
AUTHORS: ela.iwaszkiewicz, Monika Prus-Frankowska, Piotr Łukasik

[DESCRIPTION]
We have designed two synthetic spike-ins, called Callio-synth and tp53-synth, for use in metabarcoding arthropod surveys. As the starting point for designing these spike-ins, we used cytochrome c oxidase I (COI) barcode region of a blue bottle fly (Calliphora vomitoria). We preserved the primer binding sites that match primers BF3-BR2 and fwhF2-fwhR2n as well as a flanking region around each primer site (+/- 6bp). The remainder of the sequence was replaced by i) a random DNA sequence that does not resemble any known sequence deposited in GeneBank, while keeping a GC content similar to the original C. vomitoria sequence (Callio-synth spike-in); ii) short fragments of the tp53 gene (tp53-synth). What follows in this protocol are instructions on how produce and use those spike-ins in arthropod metabarcoding studies. In short, both spike-in sequences (471-bp in total) need to be synthesised and inserted into standard commercially available plasmids. They are then to be transformed into monoclonal bacteria, copied in bacteria and extracted by standard extraction kits. Same numbers of spike-in copies should then be added to every arthropod bulk sample right before the DNA extraction. Subsequently they will be co-extracted, co-amplified and sequenced together with the DNA of the sample. After sequencing, spike-in read counts can be retrieved by matching the known spike-in sequence to the ASV or OTU lists.

[BEFORE_START]
We have designed two synthetic spike-ins, called Callio-synth and tp53-synth, for use in metabarcoding arthropod surveys.

As the starting point for designing these spike-ins, we used cytochrome c oxidase I (COI) barcode region of a blue bottle fly (Calliphora vomitoria). We preserved the primer binding sites that match primers BF3-BR2 (Elbrecht et al., 2019) and fwhF2-fwhR2n (Vamos et al., 2017) as well as a flanking region around each primer site (+/- 6bp). The remainder of the sequence was replaced by i) a random DNA sequence that does not resemble any known sequence deposited in GeneBank, while keeping a GC content similar to the original C. vomitoria sequence (Callio-synth spike-in); ii) short fragments of the tp53 gene (tp53-synth).
 
 
What follows in this protocol are instructions on how produce and use those spike-ins in arthropod metabarcoding studies.
In short, both spike-in sequences (471-bp in total) need to be synthesised and inserted into standard commercially available plasmids. They are then to be transformed into monoclonal bacteria, copied in bacteria and extracted by standard extraction kits. Same numbers of spike-in copies should then be added to every arthropod bulk sample right before the DNA extraction. Subsequently they will be co-extracted, co-amplified and sequenced together with the DNA of the sample. 
After sequencing, spike-in read counts can be retrieved by matching the known spike-in sequence to the ASV or OTU lists. 

The proposed spike-ins are designed to work in experiments targeting two different primer pairs (or combination of those primers). Of these, BF3-BR2 produces a 418-bp long fragment of the Folmer region and is commonly used in Illumina-based studies, whereas fwhF2-fwhR2n allows for the amplification of a shorter fragment, only 205-bp long, which is particularly useful when dealing with degraded DNA.

Our proposed protocol was developed for homogenized Malaise trap samples (coming from the Insect Biome Atlas project; insectbiomeatlas.org) and can only be recommended for such types of samples.

[STEPS]
SECTION: Sequence synthesizing, plasmid production and transformation into E. coli
1. Download FASTA file with the spike-in sequences from GitHub: https://github.com/ela-iwaszkiewicz/Synthetic_COI_spike-ins
SECTION: Plasmid isolation from E. coli
7. The plasmids were isolated from E. coli using a GeneMATRIX PLASMID
MINIPREP (EURx) kit following the manufacturer's instructions.
SECTION: Bacterial culture
4. PREPARE LB AGAR PLATES with amplicilin anibioticum
Tryptone5 gYeast Extract2,5 gNaCl5 g Agar7,5 gWaterUp to 500 mlAmpicillin0,5ml [100mg/ml] or 1ml [50mg/ml]

You can also use ready LB-Agar mix from A&A Biotechnology (cat. no 2021-250). The proportion of the ingredients is the same. Use 4g of the LB-Agar mix per 100 ml of destilled/sterile water.
SECTION: Plasmid linealization
8. Plasmid digestion with restriction enzymes
To avoid complications during PCR that might have arisen because of circular shape pf plasmids, we decided to linearise them by cutting with a restriction enzyme.
We chose an AatII enzyme (NEB) as it was cutting the plasmid effectively and leaving the barcode region intact.

(You can always check the parameters of the protocol on the NEB cloner website http://nebcloner.neb.com/#!/)

Materials:
       Restriction enzyme AatII – we are using New England Biolabs enzymes, for the enzymes from a different manufacture the digestion protocl might be different
       CutSmart Buffer (provided with enzyme)
       Molecular water (nuclease free)

PREPARE ON ICE!
SECTION: Quantification and dilutions
9. Preparing 300 mln copies single-use aliquots
 
Materials and equipment:
       Qubit fluorometer and Qubit HS assay kit
       TE x1
       Digested plasmid
SECTION: Adding spike-ins to samples
10. We added xx ul of the plasmid DNA stock (~1M copies) of each spike-in to the sample right before DNA extraction. 

2. Use commercial services (i.e. Eurofins) to synthesise spike-in sequences and insert them onto pEX-A128 plasmid harboring ampicillin resistance.
SECTION: Long-term storage of bacteria carrying plasmids
6. GLYCEROL STOCK
For long-term storage of plamids it is adviced to create a baterial glycerol stock. 
Such stock is stable for years, as long as it kept in –80*C. 

To create glycerol stock you need:
      Overnight bacteria culture (in LB medium)
      50 % glycerol (e.g. 25 g of 100% glycerol + 25ml of dH2O)
 
[Source: https://www.addgene.org/protocols/create-glycerol-stock/]
5. Select bacteria by culturing them on agar with amplicillin
[All sub-steps in this part of the protocol are copied from  : https://www.addgene.org/protocols/inoculate-bacterial-culture/]
 
Use LB agar plates with amplicillin prepared according to the protocol in the previous step (#3).     
SECTION: Bacterial culture
4.6. Use or store the plates in 4˚C not longer than for a week. 
SECTION: Bacterial culture
4.5. Leave the plates on the bench/under the laminar to solidify – about 24 h/or overnight. 
SECTION: Bacterial culture
4.4.  Place the bootle under the hood and pour the agar into the sterile, freshly open Petri dishes (plates).
SECTION: Bacterial culture
4.3. Add the proper amount of the antibiotic stock [to obtain final concentration in media 100 ug/ml] and mix it gently. 
SECTION: Bacterial culture
4.2. After that cool the molten agar to the temperature of 55˚C.
SECTION: Bacterial culture
4.1. Mix all of the ingredients (except ampicillin), add water and autoclave it. 
5.1. Label the bottom of the plate with the plasmid name, the date and the name of antibiotic (in our case AMP).
5.2. Sterilize and turn on the hood. 
5.3.  Place fresh plates and you bacteria stock/bacteria plate under the hood. 
5.4. Using a sterile pipette tip or toothpick touch the bacteria growing within the punctured area of the stab culture or the top of the glycerol stock.
5.5. Gently spread the bacteria over a section of the plate, as shown in the diagram below, to create streak #1.

 

5.6. Using a fresh, sterile toothpick, or freshly sterilized loop, drag through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5.7. Using a third sterile pipette tip, toothpick, or sterilized loop, drag through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
5.8. Incubate plate with newly plated bacteria overnight (12-18 hours) at 37 °C.
5.9.  It is good to incubate also a blank sample (fresh plate without any bacteria on the surface). 
5.10. Prepare liguid LB with antibiotic.
5.11. Prepare fresh, sterile 15 or 50 ml centriguge tubes.

5.12. Sterilize and turn on the hood.

5.13. Transfer ca. 10ml of LB+antibiotic to 15 ml tubes (and ca. 20 ml to 50 ml tubes). Do not forget to label each tube with the date, name of the strain/plasmid.

5.14. Using a sterile pipette tip or toothpick, select a single colony from LB agar plate. You can leave the toothpick or pipette tip inside the LB.

5.15. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.

5.16. Incubate bacterial culture at 37°C for 12-18 hr in a shaking incubator.

5.17. It is good to incubate also a blank sample (fresh LB without any bacteria in it). 

5.18. After incubation you can prepare glycerol long-term stock (section 5) and/or proceed to isolating plasmid DNA (section 6).
SECTION: Long-term storage of bacteria carrying plasmids
6.1. In a 2ml screw cap tube for freezing put 500ul of overnight colony and 500ul of 50% glycerol. Mix it gently. 
SECTION: Long-term storage of bacteria carrying plasmids
6.2. Freeze the tube in –80*C. 
SECTION: Long-term storage of bacteria carrying plasmids
6.3. To recover the stock scrap a bit of the frozen bacteria from the top into a fresh
agar plate (with Ampicilin). 
SECTION: Long-term storage of bacteria carrying plasmids
6.4. Otherwise take a sample of LB medium with bacterial culture for further plasmid isolation.
SECTION: Plasmid linealization
8.1. Measure the concentration of isolated plasmid DNA on Qubit.
SECTION: Plasmid linealization
8.2.  Remove restriction enzyme and CutSmart buffer from the frezer and put it on ice. 
SECTION: Plasmid linealization
8.3. Calculate how many ul of plasmid you need to add to the reaction in order to have ca. 100 ng of plasmid DNA in each reaction. At first add molecular water to the tube, then plasmid and CutSmart buffer. As a last component add restriction enzyme. Do not vortex the reaction! Just mix it by pipetting. Centrifuge shortly.
SECTION: Plasmid linealization
8.4. Put the strip tube/plate in a termocycler and set up the program. The incubation and inactivation temepratures and time can be found in table below.   
    ABC1Restriction enzymeIncubation temp. and timeInactivation temp. and time2AatII (NEB)37˚C, 5-15 mins80˚C, 20 mins
 
SECTION: Plasmid linealization
8.5. After digestion freeze the samples or prepare 300 mln copies single-use aliquots according to the instruction below. 
SECTION: Quantification and dilutions
9.1. Measure digested plasmid DNA concentration using Qubit flurometer. 
SECTION: Quantification and dilutions
9.2. Based on the received information calculate how many copies of a plasmid you have in one ul. 
You can do this based on the assumption that 1 ng of COI plasmid DNA (as we've designed) contains 300 mln copies of the plasmid.
(This assumption is based on a calculation taking into account the size of the DNA molecule)

SECTION: Quantification and dilutions
9.3. Based on performed calculations dilute spike-in’s samples with 1xTE into the concentration of 300 mln of copies/ul and prepare single-use aliquots of 8ul each. Store in a frezer in 20˚C. 
SECTION: Quantification and dilutions
9.4. Remember that for your experiment you will need to prepare additional dilutions of a spike-ins. You can do it with molecular water (nuclease free).  
3. Transformation into E. coli
3.1. Upon receiving plasmids from your commercial supplier, perform transformation into a monoclonal bacterial strain.
In our experiment, the plasmid was cloned into JO-FI competent cells derived from the E. coli DH5α strain (A&A Biotechnology) using CloneJET PCR Clonning kit (Thermo Fisher Scientific).
3.2. A single successfully transformed colony was transferred into LB medium (A&A Biotechnology) mixed with Ampicilin Sodium (A&A Biotechnology) at the final concentration 100 ug/ml and grown overnight at 37 ̊C.