TITLE: PrimalSeq: Generation of tiled virus amplicons for MiSeq sequencing
AUTHORS: Nate Matteson, Nathan Grubaugh, Karthik Gangavarapu, Josh Quick, Nick Loman, Kristian Andersen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Generated in collaboration by the </span><a href="http://lab.loman.net" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;:UNDERLINE;">Loman</span></a><span style = "font-weight:bold;">, </span><a href="https://andersen-lab.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;:UNDERLINE;">Andersen</span></a><span style = "font-weight:bold;">, and </span><a href="http://grubaughlab.com" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;:UNDERLINE;">Grubaugh</span></a><span style = "font-weight:bold;"> labs.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">For general use of the protocol and primer design, please cite:</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Quick, J. et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. </span><span style = "font-style:italic;">Nature Protocols</span><span> 12 (6), 1261-1276 (2017)</span><a href="https://www.nature.com/nprot/journal/v12/n6/abs/nprot.2017.066.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://www.nature.com/nprot/journal/v12/n6/abs/nprot.2017.066.html</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">For measuring intrahost virus genetic diversity and calling variants using iVar, please cite:</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Grubaugh, ND. et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. </span><span style = "font-style:italic;">Genome Biology</span><span>  20,8 (2019) </span><a href="https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1618-7</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The general approach to this protocol is to amplify the virus genome in small (~400 bp) overlapping fragments using two highly multiplexed PCR reactions (where the overlapping segments are in separate reactions). The amplicons are combined after PCR and are the correct size for library preparation and paired-end 250 nt sequencing using the Illumina MiSeq.</div></div><div class = "text-block"><span style = "font-weight:bold;">Version 4 notes</span><span>: This protocol has been updated to include considerations for measuring intra-batch contamination through the introduction of sample-specific barcoded spike-ins.</span></div><div class = "text-block"><span style = "font-weight:bold;">All of the primers are now listed in a separate </span><a href="https://docs.google.com/spreadsheets/d/1zMfUv1IV5-Sy-AeOPhKHdoZMUxhD1R_RsB5gPswqx40/edit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;:UNDERLINE;">spreadsheet</span></a><span style = "font-weight:bold;">. </span><span>We currently have 400 bp amplicon schemes for Zika virus, West Nile virus (North America lineage I genotype), Usutu virus, and chikungunya virus (ECSA genotype). More will be made available soon. You can build your own primer sets by using </span><a href="http://primal.zibraproject.org" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Primal Scheme</span></a><span>. </span></div><div class = "text-block"><span style = "font-weight:bold;">Overview of tiled virus amplicon sequencing protocol</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/80ae308f781c7d59f162f5213ebeca9eec4e2590e759008ed2722b0c6cfc2d53/b5k7bct3x.png" /></div></div>

[STEPS]
?. [Generation of barcode spike-ins]
Prepare a PCR reaction for each combination of forward and reverse primers. A master mix can be created by combining all components except the forward and reverse primers.  ABC1ComponentVolume in 20 µL reactionFinal Concentration2Q5 Reaction Buffer (5x)5 µL1x3dNTPs (10 mM)0.5 µL200 uM4Forward Primer (10 µM)1 µL400 nM5Reverse Primer (10 µM)1 µL400 nM6Q5 Polymerase0.25 µL0.02 U/ul7Water16.25 µL-8Template DNA (adh1; 0.1 ng/µL)1 µL
ABC1ComponentVolume in 20 µL reactionFinal Concentration2Q5 Reaction Buffer (5x)5 µL1x3dNTPs (10 mM)0.5 µL200 uM4Forward Primer (10 µM)1 µL400 nM5Reverse Primer (10 µM)1 µL400 nM6Q5 Polymerase0.25 µL0.02 U/ul7Water16.25 µL-8Template DNA (adh1; 0.1 ng/µL)1 µL
?. [PCR generation of tiled amplicons]
Validated primer schemes can be found here. Prepare two primer pools by mixing equal volumes of each 10 µM primer. Primers indicated by “*” should be pooled at a concentration of 50 µM and primers indicated by “**” should be pooled at a concentration of 100 µM to help normalize sequencing coverage. The sequences for the primer which amplify the barcoded transcript are shown below:> Contamination Primer ForwardAATGTCGCAGGCACTTGTCC> Contamination Primer ReverseGGTCAGAGCTGTCTCCTGCTNote: Concentration for contamination primers should be identical to concentration of individual primer pairs in pool. Concentration listed below is for our West Nile virus protocol but will vary based on other schemes.
?. [Adapter ligation]
Dilute a working stock of NEXTflex Dual-Indexed DNA Barcodes 1:100 to obtain a concentration of 250 nM. Select unique barcodes for each sample. Try not to repeat barcodes from recent runs.Note: Be careful to not cross-contaminate the adaptors by centrifuging all liquid from the caps and only opening one index at a time.
?. [Library amplification]
Combine the following components:  AB1ComponentVolume in 34 µL reaction22X KAPA HiFi HotStart ReadyMix17 µL3Illumina primer mix2 µL4Adaptor-ligated library 15 µL
AB1ComponentVolume in 34 µL reaction22X KAPA HiFi HotStart ReadyMix17 µL3Illumina primer mix2 µL4Adaptor-ligated library 15 µL
?. [Generation of barcode spike-ins]
Barcode spike-ins are generated with a step-out PCR using a common insert and variable forward and reverse primers. The insert we use and structures of the variable primers is show below. X’s refer to the barcode region. We generated 8 forward barcodes, and 12 reverse barcodes which are 20bp long and have an edit distance of at least 10 nucleotides from all other generated barcodes. By mixing and matching primers a total of 96 unique barcode transcripts can be generated.> Zea mays alcohol dehydrogenase 1 (adh1), mRNA (XM_008650471.2)AGGGTCTCGGAGTGGATTGATTTGGGATTCTGTTCGAAGATTTGCGGAGGGGGGCAATGGCGACCGCGGGGAAGGTGATCAAGTGCAAAGCTGCGGTGGCATGGGAGTCCATGCAAGCCACTGTCGATCGAGGAAGTGGAGGTAGCGCCTTCGCAGGCCATGGAGGTGCGCGTCAAGATCCTCTTCACCTCGCTCTGCCACACCGACGTCTACTTCTGGGAGGCCAAGGGGCAGACTCCCGTGTTCCCTCGGATCTTTGGCCACGAGGCTGGAGGTATCATAGAGAGTGTTGGAGAGGGTGTGACTGACGTAGCTC> Barcode transcript step-out forward primer (structure)AATGTCGCAGGCACTTGTCCxxxxxxxxxxxxxxxxxxxxAGGGTCTCGGAGTGGATTGA> Barcode transcript step-out reverse primer (structure)GGTCAGAGCTGTCTCCTGCTxxxxxxxxxxxxxxxxxxxxGAGCTACGTCAGTCACACCC
?. [Generation of barcode spike-ins]
Run the following cycles on a thermocycler:  ABC1CyclesTemperatureTime2198°C30 seconds31098°C10 seconds468°C10 seconds572°C20 minutes6172°C2 minutes714°C∞
ABC1CyclesTemperatureTime2198°C30 seconds31098°C10 seconds468°C10 seconds572°C20 minutes6172°C2 minutes714°C∞
?. [Generation of barcode spike-ins]
Proceed immediately to cleanup
?. [Post PCR cleanup]
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
?. [Post PCR cleanup]
Bring PCR product volume up to 25 µL with water (if not at volume already).
?. [Post PCR cleanup]
Add 50 µL of beads to 25 µL of PCR product, mix well, and incubate at room temperature for 10 minutes.
?. [Post PCR cleanup]
Place tubes on a magnetic stand and incubate until solution appears clear.
?. [Post PCR cleanup]
Discard supernatant without disturbing the beads.
?. [Post PCR cleanup]
While tubes are on the magnet, add 200 µL of 80% EtOH, incubate for 30 seconds, and discard the EtOH wash.
?. [Post PCR cleanup]
Repeat previous 80% EtOH wash and remove as much EtOH as possible.
?. [Post PCR cleanup]
Leave tubes on magnet and air dry for 5 minutes.
?. [Post PCR cleanup]
Remove tubes from magnet and add 20 µL of nuclease-free water. Mix well by pipetting.
?. [Post PCR cleanup]
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes.
?. [Post PCR cleanup]
Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 µL of each product. Expected range = 10-100 ng/µL DNA. Sequencing from lower concentrations may still work.
?. [Post PCR cleanup]
Note: If your lab has a KingFisher, you can download our automated protocols here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this step)
?. [Preparation of cDNA]
Isolate viral RNA using Omega Viral DNA/RNA kit, Trizol, or equivalent.
?. [Preparation of cDNA]
Many different cDNA synthesis kits can be used, but choose something that is relatively high-fidelity. The current protocols uses SuperScript IV VILO Master Mix because the enzyme has low error rates and the protocol is fast and easy.
?. [Preparation of cDNA]
Dilute a working stock of Barcoded Spike-ins 1:100,000 to obtain a concentration of 38 fM. Select unique spike-in for each sample. Try not to repeat spike-ins from recent runs. Note: Be careful to not cross-contaminate the spike-ins by centrifuging all liquid from the caps and only opening one index at a time
?. [Preparation of cDNA]
AB1ComponentVolume in 20 µL reaction2SSIV VILO Master Mix4 µL3Nuclease-free water5-14 µL4Barcoded Spike-in (38 fM)1 µL5Virus RNA1-10 µL
AB1ComponentVolume in 20 µL reaction2SSIV VILO Master Mix4 µL3Nuclease-free water5-14 µL4Barcoded Spike-in (38 fM)1 µL5Virus RNA1-10 µL
?. [Preparation of cDNA]
Run the following cycles on a thermocycler:  AB1TemperatureTime225°C10 minutes350°C10 minutes485°C5 minutes54°C∞
AB1TemperatureTime225°C10 minutes350°C10 minutes485°C5 minutes54°C∞
?. [Preparation of cDNA]
Store samples at 4°C (for use same day) or -20°C (for use within a week) until ready for PCR.
?. [PCR generation of tiled amplicons]
Prepare two PCR reactions for each sample (one for each primer pool):  AB1ComponentVolume in 25 µl reaction2Q5 2x Master Mix12.5 µl3Primer pool (#1 or #2)1 µl4Contamination Primer Forward (0.26 µM)1 µl5Contamination Primer Reverse (0.26 µM)1 µl6Nuclease-free water8.5 µl7cDNA1 µl
AB1ComponentVolume in 25 µl reaction2Q5 2x Master Mix12.5 µl3Primer pool (#1 or #2)1 µl4Contamination Primer Forward (0.26 µM)1 µl5Contamination Primer Reverse (0.26 µM)1 µl6Nuclease-free water8.5 µl7cDNA1 µl
?. [PCR generation of tiled amplicons]
Run the following cycles on a thermocycler  ABC1CyclesTemperatureTime2198°C30 seconds33595°C15 seconds465°C5 minutes514°C∞
ABC1CyclesTemperatureTime2198°C30 seconds33595°C15 seconds465°C5 minutes514°C∞
?. [PCR generation of tiled amplicons]
Run 5 µl of each product on a 1% agarose gel. Each should produce a visible 400 bp band.
?. [Post PCR cleanup]
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
?. [Post PCR cleanup]
Bring PCR product volume up to 25 µL with water (if not at volume already).
?. [Post PCR cleanup]
Add 45 µL of beads to 25 µL of PCR product, mix well, and incubate at room temperature for 10 minutes.
?. [Post PCR cleanup]
Place tubes on a magnetic stand and incubate until solution appears clear.
?. [Post PCR cleanup]
Discard supernatant without disturbing the beads.
?. [Post PCR cleanup]
While tubes are on the magnet, add 200 µL of 80% EtOH, incubate for 30 seconds, and discard the EtOH wash.
?. [Post PCR cleanup]
Repeat previous 80% EtOH wash and remove as much EtOH as possible.
?. [Post PCR cleanup]
Leave tubes on magnet and air dry for 5 minutes.
?. [Post PCR cleanup]
Remove tubes from magnet and add 20 µL of nuclease-free water. Mix well by pipetting.
?. [Post PCR cleanup]
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes.
?. [Post PCR cleanup]
Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 µL of each product. Expected range = 10-100 ng/µL DNA. Sequencing from lower concentrations may still work.
?. [Post PCR cleanup]
Note: If your lab has a KingFisher, you can download our automated protocols here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this step)
?. [End-repair and A-tailing]
Combine 25-50 ng of PCR-amplified DNA from primer pool 1 and 2 together for a total of 50-100 ng in 12.5 µL (equal concentrations of each amplicon pool). QS to a total volume of 12.5 µL using nuclease-free water. Alternatively: proceed using 50-100 ng of primer pool product separately for library preparation. This allows for additional monitoring of cross-contamination. Data can be merged computationally post sequencing.
?. [End-repair and A-tailing]
Combine the following components from the Kapa Hyper prep kit for end repair:  AB1ComponentVolume in 15 µl reaction2End Repair & A-tailing buffer1.75 µl3End Repair & A-tailing enzyme mix0.75 µl4PCR-amplified DNA (50 ng)12.5 µl
AB1ComponentVolume in 15 µl reaction2End Repair & A-tailing buffer1.75 µl3End Repair & A-tailing enzyme mix0.75 µl4PCR-amplified DNA (50 ng)12.5 µl
?. [End-repair and A-tailing]
Run the following cycles on a thermocycler:  AB1TemperatureTime220°C30 minutes365°C30 minutes44°C∞
AB1TemperatureTime220°C30 minutes365°C30 minutes44°C∞
?. [Adapter ligation]
Combine the following components:  AB1ComponentVolume in 27.5 µL reaction2Ligation buffer7.5 µL3DNA ligase2.5 µL4NEXTflex DNA Barcodes (250nM)2.5 µL5End repair reaction product15 µL
AB1ComponentVolume in 27.5 µL reaction2Ligation buffer7.5 µL3DNA ligase2.5 µL4NEXTflex DNA Barcodes (250nM)2.5 µL5End repair reaction product15 µL
?. [Adapter ligation]
Incubate at 20°C for 15 minutes
?. [Adapter ligation]
Proceed immediately to cleanup
?. [Post ligation cleanup]
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
?. [Post ligation cleanup]
Add 22 µL of beads to 27.5 µL of ligation product, mix well, and incubate at room temperature for 10 minutes.
?. [Post ligation cleanup]
Place tubes on a magnetic stand and incubate until solution appears clear.
?. [Post ligation cleanup]
Discard supernatant without disturbing the beads.
?. [Post ligation cleanup]
While tubes are on the magnet, add 200 µL of 80% EtOH, incubate for 30 seconds, and discard the EtOH wash.
?. [Post ligation cleanup]
Repeat previous 80% EtOH wash and remove as much EtOH as possible.
?. [Post ligation cleanup]
Leave tubes on magnet and air dry for 5 minutes.
?. [Post ligation cleanup]
Remove tubes from magnet and add 20 µL of nuclease-free water. Mix well by pipetting.
?. [Post ligation cleanup]
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes - 15 µL will go into library amplification.
?. [Post ligation cleanup]
Note: If your lab has a KingFisher, you can download our automated protocols here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this step)
?. [Library amplification]
Run the following cycles on a thermocycler:  ABC1CyclesTemperatureTime2198°C45 seconds31298°C15 seconds460°C30 seconds572°C30 seconds6172°C1 minute74°C∞
ABC1CyclesTemperatureTime2198°C45 seconds31298°C15 seconds460°C30 seconds572°C30 seconds6172°C1 minute74°C∞
?. [Library amplification]
Proceed immediately to cleanup or store at 4°C.
?. [Post amplification cleanup]
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
?. [Post amplification cleanup]
Add 27.2 µL of beads to 34 µL of amplified product, mix well, and incubate at RT for 10 minutes.
?. [Post amplification cleanup]
Place tubes on a magnetic stand and incubate until solution appears clear.
?. [Post amplification cleanup]
Discard supernatant without disturbing the beads.
?. [Post amplification cleanup]
While tubes are on the magnet, add 200 µL of 80% EtOH, incubate for 30 seconds, and discard the EtOH wash.
?. [Post amplification cleanup]
Repeat previous 80% EtOH wash and remove as much EtOH as possible.
?. [Post amplification cleanup]
Leave tubes on magnet and air dry for 5 minutes.
?. [Post amplification cleanup]
Remove tubes from magnet and add 25 µL of Tris-EDTA or elution buffer. Mix well by pipetting.
?. [Post amplification cleanup]
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes.
?. [Post amplification cleanup]
Note: If your lab has a KingFisher, you can download our automated protocols here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this step)
?. [Library quantification and pooling]
Quantify the DNA concentration of each sample (1 µL) using the Qubit High Sensitivity DNA kit.
?. [Library quantification and pooling]
Pool equal concentrations (e.g., 1-10 ng) of each library for sequencing.
?. [Library quantification and pooling]
Check DNA fragment distributions of the pooled sample using the BioAnalyzer DNA 1000 kit. Peak fragment size from 400 bp tiled amplicons with proper ligated adaptors should be ~ 580 nt. If ~180 bp bands (adaptor dimers) still exist, perform post amplification cleanup again.
?. [Library quantification and pooling]
Quantify the DNA concentration of the pooled library (1 µL) using the Qubit High Sensitivity DNA kit.
?. [Library quantification and pooling]
Note: At least 0.76 ng/µL is required to achieve 2 nM for library pooling. Libraries will need to be concentrated or re-amplified if less than this amount.
?. [Library quantification and pooling]
Convert DNA libraries from weight to moles: Molecular weight [nM] = Library concentration [ng/µL] / ((ave. library size x 650)/1,000,000) Example: if ave. size of library is 580 bp and concentration is 2.5 ng/µL… (580 x 650) / 1,000,000 = 0.3775 / 0.377 = 6.6 nM
?. [Library quantification and pooling]
Dilute the pooled library to 2 nM in 10 mM TE.
?. [Library quantification and pooling]
(Optional) Ensure the library molar concentration using the Kapa Library Quantification kit.
?. [Library quantification and pooling]
If sending your sample to a genomics core (i.e., not loading the MiSeq yourself), stop here.
?. [Diluting the pooled library for sequencing]
Combine 10 µL of the 2nM pooled library to 10 µL of 0.1 N NaOH and mix. Incubate from 5 minutes at room temperature to denature the dsDNA.
?. [Diluting the pooled library for sequencing]
Add 980 µL of HT1 (comes with the MiSeq kits). New concentration = 20 pM.
?. [Diluting the pooled library for sequencing]
Dilute to the desired concentration using the following volumes.   ABCDE1Concentration10 pM12 pM14 pM16 pM220 pM Library295 µl255 µl415 µl475 µl3Prechilled HT1300 µl240 µl180 µl120 µl4PhiX control *5 µl5 µl5 µl5 µl*PhiX control should also be denatured and diluted to 20 pM.
ABCDE1Concentration10 pM12 pM14 pM16 pM220 pM Library295 µl255 µl415 µl475 µl3Prechilled HT1300 µl240 µl180 µl120 µl4PhiX control *5 µl5 µl5 µl5 µl
?. [Diluting the pooled library for sequencing]
Note: loading too high of a sample on a MiSeq leads to over-clustering and decreased quality, which may make the data unusable. Adding too low leads to under-clustering and may not generate enough data for sufficient sequencing coverage. In our hands, optimal cluster densities were reached using 10-12 pM with the MiSeq v2 kits and 14-16 pM with the MiSeq v3 kits. Loading concentrations should be empirically determined with each lab.
?. [Diluting the pooled library for sequencing]
Following loading instructions located in the MiSeq user guides.
?. [Data processing and analysis]
Use iVar, following instructions on: www.github.com/andersen-lab/ivar. An example pipeline for generating consensus sequences and utilizing barcode transcripts to estimate contamination can be found on: www.github.com/watronfire/PrimalSeq_Pipeline