Feb 25, 2019
Version 2
18S V4 tag sequencing PCR amplification and library prep (Illumina) V.2
- Lisa Mesrop1,
- Sarah Hu1
- 1University of Southern California
- Caron Lab - Protistan Ecology
Protocol Citation: Lisa Mesrop, Sarah Hu 2019. 18S V4 tag sequencing PCR amplification and library prep (Illumina). protocols.io https://dx.doi.org/10.17504/protocols.io.rfvd3n6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2018
Last Modified: February 25, 2019
Protocol Integer ID: 13525
Abstract
Protocol for PCR amplification of extracted cDNA or DNA and downstream library preparation for MiSeq sequencing.
See other related protocols for extraction of RNA and/or DNA from environmental samples
This protocol is specific to 18S rRNA gene tag sequencing targeting to V4 hypervariable region. We use V4 primers with Illumina adapters that then anneal to the P5 & P7 Illumina-specific indices. This barcoding step allows us to multiplex many samples for MiSeq sequencing. The V4 region is ~400bp, so we typically do MiSeq 250x250bp PE sequencing.
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
96-well Microtiter Plate Magnetic Separation RackNew England BiolabsCatalog #S1511S
AMPure XP Beads
Fresh 80% Ethanol
Thermocycler
25 ml Reservoir
Qubit 2.0 Fluorometer
Nuclease-Free Water
Mini Centrifuge
TE Buffer
V4 Illumina Adaptors
Illumina P5 & P7 Index Primers
STEP MATERIALS
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
AMPure XP Beads
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
AMPure XP Beads
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Protocol materials
Qubit 2.0 Fluorometer
Nuclease-Free Water
TE Buffer
96-well Microtiter Plate Magnetic Separation RackNew England BiolabsCatalog #S1511S
Illumina P5 & P7 Index Primers
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
V4 Illumina Adaptors
Fresh 80% Ethanol
Thermocycler
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
25 ml Reservoir
AMPure XP Beads
AMPure XP Beads
Mini Centrifuge
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
AMPure XP Beads
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
AMPure XP Beads
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Before start
- Make fresh 80% ethanol.
- Remove Agencourt AMPure XP beads from storage and let sit out at room temperature for at least 30 minutes.
- Ensure the thermocycler is preheated to Q5 activation temperature 98ºC.
- Thaw appropriate materials on ice.
Amplification step with V4 primers (optional Illumina Adaptors for in house barcoding)
Amplification step with V4 primers (optional Illumina Adaptors for in house barcoding)
We use Q5 High-Fidelity 2x Master Mix for our PCR reactions. The convenient 2x master mix formulation is easy to use and is suitable for PCR applications requiring greater accuracy and amplification of difficult or low genomic DNA concentrations. Depending on projects, we use anywhere between 1 ng to 10 ng input of DNA for a final concentration reaction mixture of .04 ng/ul to .4 ng/ul, respectively.
| Reagent | Volume | Stock Concentration | Final Concentration | |
| Q5 High Fidelity 2x Master Mix | 12.5 ul | 2x | 1x | |
| 18S V4 Forward Illumina Adaptor | 1.25 ul | 10 uM | .5 uM | |
| 18S V4 Reverse Illumina Adaptor | 1.25 ul | 10 uM | .5 uM | |
| DNA Template | Variable | |||
| Nuclease-Free Water | To 25 ul |
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Ensure the thermocycler is preheated to Q5 activation temperature 98ºC.
Note: Depending on template size, annealing and extension temperatures and times may vary. Please refer to BioLabs Protocol for Q5 High-Fidelity 2x Master Mix thermocycler conditions.
| Step | Temp | Time | Cycles | ||
| Step 1 | Activation of Q5 | 98ºC | 2 min | ||
| Step 2 | Denaturation | 98ºC | 10 sec | 10x | |
| Step 3 | Annealing | 53ºC | 30 sec | ||
| Step 4 | Extension | 72ºC | 30 sec | ||
| Repeat Steps 2 - 4 10 times | |||||
| Step 5 | Denaturation | 98ºC | 10 sec | 15x | |
| Step 6 | Annealing | 48ºC | 30 sec | ||
| Step 7 | Extension | 72ºC | 30 sec | ||
| Step 8 | Final Extension | 72ºC | 2 min | ||
| Repeat Steps 5 - 6 15 times | |||||
| Hold | 4ºC |
PCR Clean Up
PCR Clean Up
Remove Agencourt AMPure XP beads from storage and let sit out at room temperature for at least 30 minutes.
Steps 3-19 can also be found with AgencourtsAMPure bead information.
AMPure XP Beads
Make fresh 80% Ethanol.
Vortex Agencourt AMPure XP beads for 1 minute to ensure the beads are evenly distributed prior to use.
Add 1:1 volume ratio of beads to PCR product (25 ul, if you're following this protocol) into each sample and pipette mix up and down 20-30x gently.
Let mixture sit at room temperature for 10-15 minutes.
Place tubes onto magnetic stand for 2 to 5 minutes and let the beads clear from solution.
Carefully remove the supernatant without disturbing the beads.
Carefully remove the supernatant without disturbing the beads.
With the tubes on the magnetic stand, add 200 ul of freshly prepared 80% Ethanol to each sample opposite the aggregated beads being careful not to disturb them.
Incubate the tubes on the magnetic stand for 30 seconds and carefully remove the supernatant.
With the tubes on the magnetic stand, add 200 ul freshly prepared 80% Ethanol to each sample well opposite the aggregated beads being careful not to disturb them.
Incubate the tubes on the magnetic stand for 30 seconds and carefully remove the supernatant completely. Use a smaller volume pipette to remove any small amounts of ethanol collected at the bottom of the tubes.
Critical Step - With the tubes on the magnetic stand allow the beads to air-dry for 5 to 8 minutes. Make sure beads do not over dry and begin to crack.
Remove the tubes from the magnetic stand and add TE or nuclease-free water to each sample tube. Carefully pipette mix up and down for 20-30x.
Rehydrate sample tubes for 2-5 minutes.
Centrifuge sample tubes at 280 x g for 30 sec to collect solution in each tube.
Immediately place the tubes on the magnetic stand for 2-5 minutes to allow magnetic beads to collect.
Carefully transfer the supernatant to clean, labeled tubes. This is now cleaned up PCR product!
QC cleaned PCR products using Qubit fluorometer (concentration) High Specificity Dye & Buffer
- For the Qubit, load 2ul of product to 189 ul of High Specificity Dye & Buffer Solution for each sample
Indexing your PCR product with Illumina P5 & P7 indices
Indexing your PCR product with Illumina P5 & P7 indices
We have P5 & P7 Index Primers in 5 uM stocks.
Note: Make sure to normalize total input PCR product (total ng) for amplification Step 22.
| Reagent | Volume | Stock Concetration | Final Concentration | |
| Q5 High Fidelity 2x Master Mix | 12.5 ul | 2x | 1x | |
| P5 Indexed Primer | 2.5 ul | 5 uM | .5 uM | |
| P7 Indexed Primer | 2.5 ul | 5 uM | .5 uM | |
| DNA Template | Variable | |||
| Nuclease - Free Water | Up to 25 ul |
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
PCR Thermal Profile for Step 22
PCR Thermal Profile for Step 22
Ensure the thermocycler is preheated to Q5 activation temperature 98ºC.
Note: Depending on template size, annealing and extension temperature and time may vary. Please refer to BioLabs Protocol for Q5 High-Fidelity 2x Master Mix thermocycler conditions.
| Step | Temp | Time | Cycles | ||
| Step 1 | Activation of Q5 | 98°C | 2 min | ||
| Step 2 | Denaturation | 98°C | 10 sec | 8x | |
| Step 3 | Annealing | 66°C | 30 sec | ||
| Step 4 | Extension | 72°C | 30 sec | ||
| Repeat Steps 2-3 8 times | |||||
| Step 5 | Final Extension | 72°C | 2 min | ||
| Hold | 4°C |
PCR Cleanup
PCR Cleanup
Follow PCR Clean up Steps 3 to 19 (using Agencourt AMPure beads)
Quantify cleaned up PCR product with bar
Quantify cleaned up PCR product with bar
QC cleaned PCR products using Qubit fluorometer (concentration) High Specificity Dye & Buffer
- For the Qubit, load 2ul of product to 198 ul of High Specificity Dye & Buffer Solution for each sample
Manual Library Normalization & Pooling
Manual Library Normalization & Pooling
After determining the DNA concentration in ng/ul, convert this value from ng/ul to nM using the average library size of your known template (including adaptor length).
Calculate the conversion of ng/ul to nM using the following equation:
(ng/ul)*106/(660*library size)
Library Normalization & Pooling
Library Normalization & Pooling
Determine the common concentration to dilute your samples in nM. For Illumina sequencing platforms, we prefer to dilute our samples to 10 nM starting concentration. Check with your sequencing center on how they prefer to receive samples for sequencing.
Calculate the dilution of your libraries using the following equation:
(C1)(V1)=(C2)(V2)
Pipette 2 ul from each 10 nM sample into a single tube.