Bacterial 16S rrna V1-3 Amplicon Sequencing Standard Protocol

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1 Bacterial 16S rrna V1-3 Amplicon Sequencing Standard Protocol Version 1.05 Skill Prerequisites: DNA handling, gel electrophoresis, DNA concentration measurement, polymerase chain reaction (PCR) Introduction This protocol describes how to make amplicon sequencing libraries of the variable region 1 to 3 (V1-3) of the bacterial 16S rrna gene. The 16S rrna gene is used as a universal phylogenetic marker and for distinguishing and classifying bacteria. Using the 16S rrna V1-3 enables a resolution ranging from family to species level depending on the phylogenetic branches investigated. The library preparation is PCR based and relies on PCR primes targeting conserved sequence regions between the variable regions in the bacteria 16S rrna genes. This means that the analysis will not target bacteria whose sequences deviates from the consensus sequences of these conserved regions. The used primer sequences (F: 5 -AGAGTTTGATCCTGGCTCAG-3 R: 5 -ATTACCGCGGCTGCTGG-3 ) target 78.5% of the known bacteria 16S sequences with sufficient length to be evaluated with 1 or less mismatches (SILVA SSU-114 RefNR database, ). The sequenced part of the amplicon around the 16S rrna gene variable region 1 to 3 starts at E. coli position 28 and ends at E. coli position 516 and is total of 488 bp. However the actual length depends on the target species and might vary up to +/- 75 bp. The amplicon DNA molecule includes the primer sequences and the Illumina sequencing adaptors and is a total of 614 bp when amplified from E. coli and likewise smaller/larger in other species. The protocol assumes the use of 2x300 bp Illumina MiSeq reads to sequence the full V1-3 region. Materials Instruments - Nanodrop1000 (Thermo Scientific) - Electrophoresis chamber and power source - Gel-Doc - Qubit 2.0 Fluorometer (Invitrogen) and/or Infinite M1000 PRO (Tecan) - Thermocycler - MagneSphere Technology Magnetic Separation Stand (Promega) - Tapestation2200 (Agilent) Reagents/consumables - DNAse free tips (10uL, 300uL and 1000 ul) - DNAse free tubes (1.5 ml) Page 1 of 6

2 - 96-well PCR plates (VWR) - PCR strip caps - OptiPlate-96 Black (Perkin Elmer) - Nuclease Free H 2 O (Qiagen) - SeaKem LE Agarose (LONZA) - Gelred (Biotium) - 1kb Generuler - TAE-buffer 50x - Quibit - dsdna BR assay kit (Invitrogen) - QUANT-iT DNA Assay High Sensitivity kit (Invitrogen) - Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen) - dntp mix - Barcoded V1-3 adaptor mixes - Agencourt AMPure XP (Beckman Coulter) - EtOH, 99% - D1K Screentape (Agilent) Method Input sample material IMPORTANT: DNA samples must be extracted using the exact same procedure if they are to be compared. For WWTP Activated Sludge samples and Digester sludge samples use respective standard protocols available on the EB-group Google Drive. Preferably perform DNA extraction of biological triplicates and perform independent amplicon analysis of on each replicate. Sample DNA QC and dilution 1. Quality and Concentration Check a. Nanodrop DNA measurement. Use guidelines provided by the vendor. A260/280 should be 1.8 to 2.0. A260/ to 2.2. Also compare the UV-vis absorbance curve to that of pure DNA. NB: DNA extracted with FastDNA spin kit for soil is known to contain contamination producing a peak around 230 nm. The contaminant does not effect this protocol but may result in overestimation of the DNA concentration. 2. Preliminary dilution 1. Based on the Nanodrop concentrations measurements dilute all samples to 20 ng/ul and in volume of at least 15 ul in a 96-well plate using DNA H 2 O. 3. Run gel electrophoresis (optional) 1. Prepare a 1% agarose gel 1. 1 g agarose, 100 ml 1xTAE-buffer, heat in microwave and gently swirl to completely dissolve agarose. After slight cooling add 2 ul gelred and cast gel. 2. Prepare samples by mixing 1 ul 6x loading buffer, 2 ul sample (20-50 ng) and 3 ul DNA H 2 O. Prepare ladder according to vendor recommendations. 3. Run electrophoresis approximately 80 min at 120V (6 V/cm) 4. Capture gel image on gel-doc, save and analyze. The majority of DNA should be in the range kb. If it is below the DNA is heavily degraded and it might affect the analysis. Page 2 of 6

3 4. Measure DNA concentration of diluted samples (ca. 20 ng/ul) using either Qubit/TECAN 1. Quant-iT HS DNA Assay Kit/Quibit dsdna HS assay kit: Follow standard protocol. Recalibrate instrument with standards every time. Measure samples and standards in duplicate. Load at least 2 ul sample to minimize pipetting error. Re-measure samples where the duplicate values are >10% different from their average. 5. Based on Quant-iT/Quibit measurements dilute to 5 ng/ul with DNA H 2 O. Library PCR 1. Library PCR reaction is run in duplicate for each sample. Remember negative control (DNA H2O) and positive control (sample run before). 2. Prepare master mix: Reagents Final conc. 1 reaction [µl] DNA H2O x10 buffer Platinum High Fidelity x1 2.5 dntp (5 mm) 400 µm 2 MgSO4 (50mM) 1.5 mm 0.75 Platinum Taq DNA Polymerase High Fidelity (0.5 U/µl) 2mU 0.1 Total volume Transfer 21 ul of mastermix to the wells of a 96-well PCR plate. 4. Add 2 ul of assigned barcoded V1-3 adaptor mix (5uM) to each tube. Final conc. is 400 nm. 5. Add 2 ul of template (ca. 10 ng total DNA) 6. Run PCR program 16S AMP PCR V13 30C Temp Time 95 C 2 min 30 cycles 95 C 20 sec 56 C 30 sec 72 C 60 sec 72 C 5 min Page 3 of 6

4 4 C Forever 7. Pool the duplicates. Library QC 1. DNA concentration i. Quant-iT HS DNA Assay Kit/Quibit dsdna HS assay kit: Follow standard protocol. Recalibrate instrument with standards every time. Measure samples and standards in duplicate. Load at least 2 ul sample to minimize pipetting error. Re-measure samples where the duplicate values are >10% different from their average. ii. Remember to measure the concentration of the blank sample. Subtract this reference concentration from all other samples. Use this to detect possible failed PCR reactions. 2. Gel electrophoresis i. Based on the DNA concentrations pick all the potentially failed PCR reactions, 3 good libraries and the negative and positive PCR sample. ii. iii. Library Pooling Run Tapestation 2200 with D1K Screentapes. Follow standard protocol. No replicates. Insure that target amplicon is present (614 bp) and the most abundant product and that no contaminating peaks are present at >400 bp. Depending on the PCR efficiency there can be significant contamination present <400 bp. 1. Pool libraries in equimolar concentrations. The simplest way is to calculate how much sample volume is needed to obtain a specific amount of library. ie. sample concentration is 5 ng/ul and 10 ng library DNA is wanted from each sample = 2 ul sample. Remember another sample might have a concentration of 2.5 ng/ul sample which means 4 ul sample is needed. Library Pool Cleanup 2. Gently shake Agencourt AMPure XP Bottle to resuspend beads, remove required amount and let it equilibrate to room temperature. 3. Transfer 40 ul of bead solution to a 1.5 ml spintube, add 50 ul of library pool to the beads and mix with pipette (10 times up and down). 4. Incubate for 5 minutes at room T. 5. Place sample spintube in magnetic rack for spintubes for 2-4 minutes, until liquid is cleared. 6. Remove liquid with pipette and discard it. 7. Wash bead-pellet with 200 ul 70% EtOH by gently aspirating it over the beads with a pipette. Let it rest for 30s and then remove. Repeat once. NB: EtOH solution should be made fresh every time 8. Ensure no excess liquid is left after the washes. If there is, remove it with a 10 ul pipette. 9. Dry for approximately 10 min to evaporate the EtOH NB: Longer dry times will dry out the pellet and make it difficult to resuspend, resulting in product loss. 10. Remove Place sample spintube from magnetic rack. Add 23 ul of DNA H 2 O water to each well and mix with pipette (>10x up and down) to resuspend beads. 11. Place sample spintube back on the magnetic rack and wait until the liquid clears. 12. Transfer 20 ul of the liquid to a new spintube. Page 4 of 6

5 Library Pool QC 1. DNA concentration (see above) 2. Gel electrophoresis (see above) i. Only the target amplicon (614 bp) should be present on the gel. Literature Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., Knight, R. (2011). Global patterns of 16S rrna diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl, doi: /pnas Caporaso, J. G., Lauber, C. L., Walters, W. a, Berg-Lyons, D., Huntley, J., Fierer, N., Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME journal, 6(8), doi: /ismej HMP Consortium - 16S 454 Sequencing Protocol, Version 4.2.2, Effective Date: October 27, 2010 Authors: Jumpstart Consortium Human Microbiome Project Data Generation Working Group Page 5 of 6

6 Revision History V Protocol was created on the basis of numerous journals. V Elaborated on the planning step. Elaborated on DNA input requirements Gel check of DNA quality o Corrected Agarose concentration from 1.0% to 0.5%. o Changed the target DNA molecular size to 3-10 kb. This is the range we see when using our standard DNA extraction protocol (4x40s at 6 m/s bead beating with the FastSPIN DNA kit for soil). Measure DNA Concentration of Diluted samples o Changed replicates from triplicates to duplicates. Our experience have shown that there are little variance on the DNA concentrations measurements performed with primarily the TECAN but also to some degree the Quibit. Furthermore we believe that an exact DNA concentrations is not critical at this point. If it s off <30% it should be okay, but strictly this has not been proven. By reducing from triplicates to duplicates we use less reagents and save time. Mastermix o Corrected dntp stock concentration and final concentration. o Small corrections Library Cleanup o Added 96-well plate ref. number o Small corrections Measure DNA Concentration of libraries o See above Draft Chapter for Storage and reporting o We need to think more about proper naming. V Added prerequisites for using the protocol Introduction o Added an introduction to the protocol, which elaborates a bit on the target region of the 16S. Minor corrections V Minor corrections Cleaned up sample submission description. Added list of materials V Minor corrections Library Cleanup step 2: Changed amount of Ampure bead solution used from 90 ul to 40 ul. This change insures a more stringent removal of unspecific products of up to around 200 bp. At the same time the cost is reduced. V Minor corrections and updates to the introduction. Major changes to library clean up, pooling and validation. Instead of cleaning up all samples and then performing pooling, we perform pooling before cleanup. This should save time and expensive reagents. It should still be possible to detect poor libraries before pooling with the concentration measurement and the gel electrophoresis. Updates to the submission procedure Page 6 of 6

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