RGG pooling, cleaning and library preparation protocol

Research Group Genetics Metabarcoding protocol: Pooling and Library Preparation for sequencing This setup is for sequencing amplicons on Illumina platforms NB always use in combination with the metabarcoding library preparation. Checklist. 1) After the PCR get someone to pack your plates in 2 plastic bags, and pass it to you in the semiclean library preparation lab. 2) Throw away the outer bag and clean with bleach the inner bag around your samples. 3) Put your samples in the fridge (if not working straight away) otherwise place on the lab bench to equilibrate to room temperature while you clean the hoods and equipment following the protocol. 4) Take necessary chemicals out of the freezer 30 mins prior to work to defrost. This takes approx. the same time as UV’ing the hood after cleaning, so do this simultaneously. a. Ampure beads b. ETOH (in freezer) c. Water (fridge) d. Standards for qubit BR Pooling and min elute 1. For each plate use a multichannel pipette to pool 15 microliters of each sample into an eightwell strip. 2. Combine the 8 well strip into an 1.5ML LOBIND eppendorf tube, label this with the plate name, marker (e.g. COI, 12S), pre-pool, your name and the date. 3. For each pool make 6 new eppendorf tubes, each with 125microL of the pool and add 625microL of PB buffer (minelute kit). 4. Vortex and briefly spin down the 6 tubes. 5. Prepare 3 minelute spin columns, and add the 750microL sample PB buffer mix to these columns. 6. Spin the column at 13 RPM for 1 minute 7. Throw away the flowthrough and then add the remaining 3 tubes of sample PB mix to these 3 minelute columns. 8. Spin the column at 13 RPM for 1 minute 9. Throw away the flowthrough and then add 750 microL of PE buffer to the columns 10. Spin the column at 13 RPM for 1 minute 11. Throw away the flowthrough 12. Spin the column at 13 RPM for 1 minute 13. Add 15microL of EB buffer to each column making sure that it only touches the filter. 14. Spin the column at 13 RPM for 1 minute Authors: Owen S. Wangensteen Marta Turon Julie Bitz-Thorsen Melissa Brandner Kim Præbel (kim.praeb[email protected]) BFE/NFH - Research Group for Genetics Last updated: January 2025 PO Box 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmotta[email protected] / uit.no / org. no. 970 422 528 2 15. Combine the 3 tubes with 15 microliters into one DNA lo-bind tube and label with the plate name, marker (e.g. COI, 12S), post-pool, your name and the date. Quantification 1. Make a qubit Broad range master mix by combining 1 microliter dye with 99 microliter buffer for x = n+2 samples (n= number of samples+ number of standards) a. e.g. 10 samples n=12 x= 14 b. Dye = 14 micro L. Buffer = 2786 microL 2. Add 198 microL of master mix (MM) to 10 sample tubes and 190 microL of MM to 2 standard tubes. 3. Add 2 microL of each plate post pool to each qubit sample tube. 4. Add 10 microL of each standard to each qubit standard tube. 5. Start up the qubit fluorometer , select dsDNA broad range and measure the standards first then the samples. Library preparation This protocol is modified for the Qiaseq one step amplicon library preparation kit Cleanup = Ampure beads (BIOO method) Start DNA concentration 3000 ng 1. Prepare either an 8 well strip or plate dependent on the number of libraries to prepare. 2. Combine the following in each well: Component Volume/reaction (microL) DNA (post pool minelute) 3000ng X water 31.5-X 4x 1-step Library Buffer 12.5 Y adapter 4 1-step Amplicon enzyme mix 2 Total 50 3. Set pipete to 20-30 microL mix the components by pipetting up and down 10 times 4. Program a thermal cycler to incubate at 25C for 1 hour. Cleanup Cleanup BIOO PCR free 30 minutes 1. Measure each sample using a 100microL pipette set to 50 microL 2. Make sample up to 54.5 microL with H2O 3. Add 44 microL beads and pipette up and down 10 times to mix PO Box 6050 Langnes, N-9037 Tromsø / 77 64 40 00 / postmotta[email protected] / uit.no / org. no. 970 422 528 3 4. wait 5 minutes 5. Place the tubes on the magnetic rack for 2 minutes 6. Remove and discard supernatant 7. Wash 2 x with 80% ETOH (freshly prepared) 200 microL 8. Dry 3-5 mins 9. Resuspend with 57microL resuspension buffer 10. Wait 2 mins, place on stand and transfer supernatant to a clean tube 54.5 microL 11. Add 44 microL of beads 12. Incubate 5 minutes then place on magnet 13. Remove supernatant and discard 14. Wash 2 x with 80% ETOH 15. Dry 3-5 mins 16. Add 22.5 MicroL resuspension buffer 17. Incubate 2 minutes 18. Place on a magnet for 5 minutes 19. Transfer 20 microL of library 20.