TITLE: Synthetic COI spike-ins for use in metabarcoding-based insect biodiversity surveys 
AUTHORS: Ela Iwaszkiewicz-Eggebrecht, Monika Prus-Frankowska, Piotr Łukasik

[DESCRIPTION]
In the context of insect monitoring, metabarcoding has emerged as a pivotal method, revolutionizing the precision and scope of surveillance efforts. This technique enables rapid and accurate identification of insect species from environmental samples, providing crucial insights into biodiversity, population dynamics, and ecological interactions. While metabarcoding stands out as a powerful tool, numerous challenges persist within the field. The considerable variation in methods, procedures, and protocols across studies introduces complexity and hinders seamless comparisons between datasets. The lack of standardization poses a significant obstacle, emphasizing the need for concerted efforts to establish uniform practices. To address this gap, a viable strategy involves the design and implementation of universal internal standards, such as synthetic spike-ins. Spike-ins serve as sample-specific internal positive controls as well as facilitate standardisation of the results between samples and experiments. Here, we propose two synthetic COI spike-ins designed to be used in metabarcodin-based insect biodiversity surveys. The protocol provides spike-in sequences and a detailed step-by-step description of the procedures involved in production, usage and long-term storage of those spike-ins.

[BEFORE_START]
This protocol describes the use of two synthetic spike-ins, called Calli-synth and tp53-synth, designed for application in metabarcoding-based arthropod surveys.

Spike-ins provide sample-specific internal positive control. They also facilitate between-samples normalisation procedures. By adding the same number of spike-in copies to each sample, sequencing results can be standardized, revealing meaningful differences between samples.

Short description of the spike-in design:
The barcode region of the cytochrome c oxidase I (COI) of a blue bottle fly (Calliphora vomitoria) was selected as the foundational template. We preserved the sequence of the primer binding sites, corresponding to primers BF3-BR2 (Elbrecht et al., 2019) and fwhF2-fwhR2n (Vamos et al., 2017), along with the inclusion of a flanking region around each primer site (+/- 6bp). The remaining sequence - in between the preserved sites - was modified in two distinct ways:

i) Introduction of a random DNA sequence devoid of any resemblance to known sequences deposited in GenBank. This novel sequence was carefully crafted to maintain a GC content similar to the original C. vomitoria sequence. This formed the Calli-synth spike-in sequence.

ii) Integration of short, randomly chosen, fragments from the tp53 gene, shaping the tp53-synth spike-in sequence.

 
 
Contents of the protocol:
In this protocol we outline detailed instructions for the synthesis and application of these spike-ins in arthropod metabarcoding studies.

In brief, the two spike-in sequences, totaling 471 base pairs each, are first synthesized and inserted into standard commercially available plasmids (commercial service). Subsequently, the plasmids are transformation into monoclonal bacteria, followed by amplification in bacterial culture. The amplified plasmids are then selectively grown on an antibiotic medium and extracted using standard plasmid extraction kits.
Post-extraction, the plasmids can be linearized and quantified. Subsequently, appropriate spike-in dilutions are prepared based on the DNA concentration. The primary objective is to add an equal number of spike-in copies to each arthropod bulk sample immediately before DNA extraction.
These spike-ins are then co-extracted, co-amplified, and co-sequenced alongside the sample's DNA, ensuring a unified and consistent approach throughout the process.



[STEPS]
SECTION: Sequence synthesizing, plasmid production and transformation into E. coli
1. Download FASTA file with 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 protocol 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 aim to add 5 mln of EACH plasmid copies to each sample right before DNA extraction. In order to achieve this we prepare fresh dilutions of the plasmid stock prepared in the previous section.

NOTE: Please bear in mind that 5 mln of copies of each plasmid is what we found and appropriate amount for homogenate bulk insect samples (originating from the Insect Biome Atlas project - insectbiomeatlas.org), where we expected relatively high DNA yields. 
***You must consider adjusting the numbers of copies of spike-ins for your project needs.***
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 μg/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. 10 ml 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 500 μl of overnight colony and 500 μl 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 thermal cycler 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 μl. 
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/μl and prepare single-use aliquots of 8 μl each. Store in a frezer in 20˚C. 
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 μg/ml and grown overnight at 37 ̊C.
SECTION: Adding spike-ins to samples
10.1. Thaw one single-use aliquot of the spike-in (prepared in step 9.3). Measure DNA concentration with qubit to ensure accurate calculations later on (we expect ~1 ng/μl, however we know that small DNA particles stick to walls of plastic and are being lost in crevices).
SECTION: Adding spike-ins to samples
10.2. Based on the measurement transfer 4 ng of the plasmid DNA (~ 4μl of the aliquot) to a new 1.5ml Eppendorf tube. Fill it up to 500 μl with water or TE buffer. This is your working stock.

The working stock of the spike-in should have a DNA concentration of 0.008 ng/ml.
SECTION: Adding spike-ins to samples
10.4. Throw away what is not used up within a few hours.
Prepare new working stock (repeat steps 10.1-10.4) when needed.
SECTION: Adding spike-ins to samples
10.3. Add 2 μl of this working stock to each homogenate sample right before the DNA extraction step.
This is ~ 5 mln copies of the plasmid containing spieke-in DNA.