TITLE: 18S V4 tag sequencing PCR amplification and library prep (Illumina)
AUTHORS: Lisa Mesrop,  Sarah Hu

[DESCRIPTION]
<p> </p>
<p>Protocol for PCR amplification of extracted cDNA or DNA and downstream library preparation for MiSeq sequencing.</p>
<p> </p>
<p>See other related protocols for extraction of RNA and/or DNA from environmental samples</p>
<p> </p>
<p>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 &amp; 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. </p>

[STEPS]
?. [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.   ABCD1Reagent&nbsp;Volume&nbsp;Stock Concentration&nbsp;Final Concentration2Q5 High Fidelity 2x Master Mix&nbsp;12.5 ul2x&nbsp;1x&nbsp;318S V4 Forward Illumina Adaptor&nbsp;1.25 ul&nbsp;10 uM.5 uM418S V4 Reverse Illumina Adaptor&nbsp;1.25 ul&nbsp;10 uM&nbsp;.5 uM5DNA Template&nbsp;Variable&nbsp;&nbsp;6Nuclease-Free Water&nbsp;&nbsp;To 25 ul&nbsp;&nbsp;
ABCD1Reagent&nbsp;Volume&nbsp;Stock Concentration&nbsp;Final Concentration2Q5 High Fidelity 2x Master Mix&nbsp;12.5 ul2x&nbsp;1x&nbsp;318S V4 Forward Illumina Adaptor&nbsp;1.25 ul&nbsp;10 uM.5 uM418S V4 Reverse Illumina Adaptor&nbsp;1.25 ul&nbsp;10 uM&nbsp;.5 uM5DNA Template&nbsp;Variable&nbsp;&nbsp;6Nuclease-Free Water&nbsp;&nbsp;To 25 ul&nbsp;&nbsp;
?. 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.   ABCDE1&nbsp;Step&nbsp;TempTime&nbsp;Cycles&nbsp;2Step 1Activation of Q5&nbsp;98ºC&nbsp;2 min&nbsp;3Step 2Denaturation&nbsp;98ºC10 sec&nbsp;10x4Step 3&nbsp;Annealing&nbsp;53ºC30 sec&nbsp;5Step 4&nbsp;
Extension&nbsp;72ºC30 sec&nbsp;6Repeat Steps 2 - 4 10 times&nbsp;
7Step 5&nbsp;Denaturation98ºC10 sec&nbsp;15x &nbsp;&nbsp;8Step 6&nbsp;Annealing&nbsp;48ºC30 sec9Step 7&nbsp;Extension&nbsp;72ºC30 sec&nbsp;10Step 8&nbsp;Final Extension&nbsp;72ºC2 min&nbsp;11Repeat Steps 5 - 6 15 times&nbsp;&nbsp;12&nbsp;Hold&nbsp;4ºC&nbsp;&nbsp;
ABCDE1&nbsp;Step&nbsp;TempTime&nbsp;Cycles&nbsp;2Step 1Activation of Q5&nbsp;98ºC&nbsp;2 min&nbsp;3Step 2Denaturation&nbsp;98ºC10 sec&nbsp;10x4Step 3&nbsp;Annealing&nbsp;53ºC30 sec&nbsp;5Step 4&nbsp;
Extension&nbsp;72ºC30 sec&nbsp;6Repeat Steps 2 - 4 10 times&nbsp;
7Step 5&nbsp;Denaturation98ºC10 sec&nbsp;15x &nbsp;&nbsp;8Step 6&nbsp;Annealing&nbsp;48ºC30 sec9Step 7&nbsp;Extension&nbsp;72ºC30 sec&nbsp;10Step 8&nbsp;Final Extension&nbsp;72ºC2 min&nbsp;11Repeat Steps 5 - 6 15 times&nbsp;&nbsp;12&nbsp;Hold&nbsp;4ºC&nbsp;&nbsp;
?. [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.
?. Vortex Agencourt AMPure XP beads for 1 minute to ensure the beads are evenly distributed prior to use.
?. Make fresh 80% Ethanol.
?. 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. ]
?. 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
?. [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.   ABCD1Reagent&nbsp;Volume&nbsp;Stock Concetration&nbsp;Final Concentration &nbsp;2Q5 High Fidelity 2x Master Mix&nbsp;12.5 ul&nbsp;2x &nbsp;1x&nbsp;3P5 Indexed Primer&nbsp;2.5 ul&nbsp;&nbsp;5 uM.5 uM4P7 Indexed Primer&nbsp;2.5 ul&nbsp;5 uM.5 uM5DNA Template&nbsp;Variable&nbsp;&nbsp;&nbsp;6Nuclease - Free&nbsp;Water&nbsp;Up to 25 ul&nbsp;&nbsp;&nbsp;
ABCD1Reagent&nbsp;Volume&nbsp;Stock Concetration&nbsp;Final Concentration &nbsp;2Q5 High Fidelity 2x Master Mix&nbsp;12.5 ul&nbsp;2x &nbsp;1x&nbsp;3P5 Indexed Primer&nbsp;2.5 ul&nbsp;&nbsp;5 uM.5 uM4P7 Indexed Primer&nbsp;2.5 ul&nbsp;5 uM.5 uM5DNA Template&nbsp;Variable&nbsp;&nbsp;&nbsp;6Nuclease - Free&nbsp;Water&nbsp;Up to 25 ul&nbsp;&nbsp;&nbsp;
?. [PCR Cleanup]
Follow PCR Clean up Steps 3 to 19 (using Agencourt AMPure beads)
?. [Quantify cleaned up PCR product with bar]
QC cleaned PCR products using Qubit fluorometer (concentration) High Specificity Dye & Buffer
?. [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)
?. [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.   ABCDE1&nbsp;Step&nbsp;TempTimeCycles2&nbsp;Step 1&nbsp;Activation of Q5&nbsp;&nbsp;98°C&nbsp;2 min&nbsp;&nbsp;3&nbsp;Step 2Denaturation&nbsp;98°C&nbsp;10 sec&nbsp;8x&nbsp;&nbsp;4&nbsp;Step 3&nbsp;Annealing&nbsp;66°C&nbsp;30 sec5&nbsp;Step 4&nbsp;Extension72°C&nbsp;30 sec&nbsp;6Repeat Steps 2-3 8 times7&nbsp;Step 5Final Extension&nbsp;&nbsp;72°C&nbsp;2 min&nbsp;&nbsp;8&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;
ABCDE1&nbsp;Step&nbsp;TempTimeCycles2&nbsp;Step 1&nbsp;Activation of Q5&nbsp;&nbsp;98°C&nbsp;2 min&nbsp;&nbsp;3&nbsp;Step 2Denaturation&nbsp;98°C&nbsp;10 sec&nbsp;8x&nbsp;&nbsp;4&nbsp;Step 3&nbsp;Annealing&nbsp;66°C&nbsp;30 sec5&nbsp;Step 4&nbsp;Extension72°C&nbsp;30 sec&nbsp;6Repeat Steps 2-3 8 times7&nbsp;Step 5Final Extension&nbsp;&nbsp;72°C&nbsp;2 min&nbsp;&nbsp;8&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;
?. [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)
?. [Library Normalization & Pooling]
Pipette 2 ul from each 10 nM sample into a single tube.
?. [Carefully remove the supernatant without disturbing the beads. ]
?. [Quantify cleaned up PCR product with bar]