Jan 09, 2026

Public workspaceJCVSeq

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l21w14g1y/v1
  • Ellie Bourgikos1,
  • Mallery I Breban1,
  • Nicole M Feriancek1,
  • Verity Hill1,
  • Chantal Vogels1,
  • Nathan Grubaugh1
  • 1Department of Epidemiology of Microbial Diseases, Yale School of Public Health
Ellie Bourgikos
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l21w14g1y/v1
Protocol Citation: Ellie Bourgikos, Mallery I Breban, Nicole M Feriancek, Verity Hill, Chantal Vogels, Nathan Grubaugh 2026. JCVSeq. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21w14g1y/v1
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: November 27, 2025
Last Modified: January 09, 2026
Protocol Integer ID: 233667
Keywords: segments of the jcv genome, jcv genome, jcv diversity across lineage, specific small segment utr primer, segment specific small segment utr primer, large utr primer, jcv diversity, genome coverage, multiplexed pcr primer, separate primer set, developed jcv segment, primer design, multiplexed approach to primer design, sequencing protocol, jcv segment, specific primer, jcvseq in order, jcvseq, lineage, primer, bp amplicon, final primer, pcr
Abstract
In order to capture all three segments of the JCV genome, we employed a multiplexed approach to primer design by combining previously developed JCV segment-specific primers and an internal set generated by PrimalScheme. To maximize genome coverage, we developed our own highly multiplexed PCR primers for the medium and large segments using PrimalScheme. Based on publicly available sequences, we chose to make two separate primer sets for the highly divergent A and B lineages, each forming 800 bp amplicons. Because the A and B primers share identical binding sites, they can be mixed together by pool during the sequencing protocol to cover JCV diversity across lineages. The final primer set consists of segment specific small segment UTR primers, segment specific medium and large UTR primers, and internal A and B lineage primers for the medium and large segments.
Guidelines
It is recommended that steps performed up to amplicon generation be performed at a different workstation and with different equipment than steps post-amplicon generation.
Materials
Equipment
  • 96-well format Thermocycler (two needed for 49+ sample runs)
  • Qubit
  • Bioanalyzer
  • Magnetic rack fit for a 96-well PCR plate
  • Magnetic rack fit for 1.5 / 2-mL microcentrifuge tubes
  • Cold block fit for a 96-well PCR plate
  • Pipettes (assorted sizes)
Consumables
  • 8-strip PCR tubes; may be substituted with PCR plates with heat-sealing film
  • 1.5 / 2mL / 5 mL microcentrifuge tubes
  • Filtered pipette tips (assorted sizes)
  • Reservoirs
  • Waste containers
Reagents


Bioinformatics pipeline
The Grubaugh Lab's virus agnostic analysis pipeline can be adapted to perform consensus assembly and generate quality control plots for JCVSeq. The pipeline can be found here: https://github.com/grubaughlab/DENV_pipeline. To run for this primer scheme and assemble Jamestown Canyon virus genomes, replace reference and BED files with the following files.


Reference genomes
Download JCVSb.fastaJCVSb.fasta0B
Download JCVSa.fastaJCVSa.fasta0B
Download JCVMb.fastaJCVMb.fasta4KB
Download JCVMa.fastaJCVMa.fasta4KB
Download JCVLb.fastaJCVLb.fasta6KB
Download JCVLa.fastaJCVLa.fasta6KB

BED files
Download JCVSb.bedJCVSb.bed0B
Download JCVSa.bedJCVSa.bed0B
Download JCVMb.bedJCVMb.bed0B
Download JCVMa.bedJCVMa.bed0B
Download JCVLb.bedJCVLb.bed1KB
Download JCVLa.bedJCVLa.bed1KB

Troubleshooting
qPCR
Briefly vortex and centrifuge all reagents.
Prepare 10 µM working stocks of the primers and probes below, by adding 10 µL of 100 µM stock to 90 µL of nuclease-free water.
Primer/Probe NameSequence
CAES_JCV154_FTAATGCAGCAAAAGCCAAAG
CAES_JCV307_RAAGCCGATGGATGGTAAGAT
CAES_JCV181_PCGCTCGTAAACCGGAGCGGA
qPCR primers developed by the Connecticut Agricultural Experiment Station (CAES) as part of unpublished JCV research

On ice, prepare a master mix containing the following:
Reagent NameVolume per Sample
Luna Master Mix10 µL
RT1 µL
CAES_JCV154_F1 µL
CAES_JCV307_R1 µL
CAES_JCV181_P0.5 µL
Nuclease Free Water1.5 µL

Place the 96-well PCR plate on a cold block and add 15 µL of PCR master mix to each designated well.
Add 5 µL of RNA to each designated well. Mix by pipetting, avoiding bubbles.
Add 5 µL of positive control and non-template control to the final two wells. Mix by pipetting, avoiding bubbles.
Our recommended positive control is a gene block targeting the small segment. Its sequence is:
NameSequence
JCVS_gBlockCAGATGCAGGGTTTGTGGCATTTATGGCTGACCATGGGGAATCTGTCAGTCTGTCAGCCGTTAGGATCTTCTTCCTTAATGCAGCAAAAGCCAAGGCTGCTCTCGCTCGTAAACCGGAGCGGAAAGCTACTCCTAAATTTGGAGAGTGGCAGGTGGAAATTGTCAATAATCATTTTCCTGGAAACAGGAACAACCCAATTGGTAACAACGATCTTACCATCCATCGGCTTTCAGGATATCTAGCTAGATGGGTTCTTGAGCATTTTACTACAGATGATGATGAGTCCCAGAGAGAACTCATAAGGAGCACCATCATTAATCCAATTGCAGAGTCCAATGGCATTCATTGGAACAATGGCCCAGAGATTTATCTTTCATTCTTTCCAGGAACAGAAATGTTTTTGGAAATTTTCAAATTCTATCCCTTGACCATTGGAATTTACAGAGTCAAGCATGGTATGATGGACCCTCAGTATCTGAAGAAGGCTCTCAGACAGCGC

Seal with a transparent plastic PCR seal. Centrifuge in the plate spinner.
Set the thermocycler to read FAM.
Run the following thermocycler conditions:
TemperatureTimeCycles
55 ºC10 minutes
95 ºC1 minute44 cycles
95 ºC10 seconds(included in above 44)
60 ºC30 seconds(inclued in above 44)
READ PLATE

Primer Pooling and Preparation
Combine the following primers at equal volume into a 20 µM pool. This will be AB1, containing pool 1 of primers covering diversity for both major lineages A and B.
Primer NameSequence
NYS_JCVSMF1AGTGTACTACCAAGTATAGAAAACGTTCA
NYS_JCVSM991R1AGTAGTGTGCTCCACTGAATACATTTAA
NYS_JCVMF1AGTAGTGTACTACCAAGTATAGAAAACGTT
Yale_JCVM837R1BGGGTGATAAACTAACCCGCACA
Yale_JCVM837R1AGGGTGATACACCAAACCACATAGT
Yale_JCVM1257F3BGTGATATGTATCATGAAAAAGCCGGT
Yale_JCVM1257F3AGTGACATGTACCATGAAAAAGCTGG
Yale_JCVM2057R3BTGTCTTTTCGGGCCATAGCTTC
Yale_JCVM2057R3ATATCTTTGCGAGCCATAGCTTCTG
Yale_JCVM2611F5BTGGGAACTGTGATGTTCAAGAAAATG
Yale_JCVM2611F5ATGGAAATTGCAATGTTCAAGAAAATGATT
Yale_JCVM3440R5BTGGGAGTTATAGCATTTAAGTTTGTGC
Yale_JCVM3439R5AAGGAGTTACAGCATTTAAATTAGTGCAGTA
Yale_JCVM3680F7BGACAACGATTACCAAGCTTGCAA
Yale_JCVM3680F7AGACAATGATTATCAAGCTTGCAAATTTCT
NYS_JCVM4510R2AGTAGTGTGCTACCAAGTATATCTAAATGA
NYS_JCVLF1AGTAGTGTACTCCTATTTACAAAACTTACAAATAC
Yale_JCVL873R1BGGGCTGTTTGTAATCTCCAGTGG
Yale_JCVL873R1AGGGCTGTTTGTAGTTCCCTGT
Yale_JCVL1177F3AGCTGGCGACAAGTTATGAATAAGA
Yale_JCVL2014R3BGTGCAGGCTCAGTTAGAGATAACA
Yale_JCVL2014R3AGTGCAGGCTCAGTTAAGGACAA
Yale_JCVL2315F5BCCAAAGGTCTGCATGAGAAGCA
Yale_JCVL2315F5ACTAAGGGTTTACATGAGAAACATCATGTT
Yale_JCVL3121R5BCCCTATTTTTTTGCCTAGTAGTTTCAACT
Yale_JCVL3121R5ACCCTATTCTTTTGTCTTGTAGTCTCAAC
Yale_JCVL3638F7BCAATAGTTCAAGATAAGGCCCCTGA
Yale_JCVL3638F7ACGATAGTTCAAGACAAGGCTCCA
Yale_JCVL4432R7BCATTCATGCCTCCTGGTGATGATA
Yale_JCVL4432R7ACATTCATTCCTCCTGGTGATGACA
Yale_JCVL4975F9BGCAGACCCAACAGAGATGTCAA
Yale_JCVL4975F9AGCTGATCCAACAGAGATGTCAAGA
Yale_JCVL5804R9BGCCATATTTTCGAATTTTAGACCATGC
Yale_JCVL5804R9AGCCATATTTTCAAATTTCAAGCCATGT
Yale_JCVL6087F11BAAAGAAAGCACATTTTAGCAAAATGGTATC
Yale_JCVL6087F11AAAAGAAGGCGCATTTCAGCAAAA
NYS_JCVL6957R2AGTGTGCTCCTATTTACAAATATATACTATAAGC
Primers developed using PrimalScheme, in collaboration with New York State Wadsworth Center

Combine the following primers at equal volume into a 20 µM pool. This will be AB2, containing pool 1 of primers covering diversity for both major lineages A and B.
Primer NameSequence
Yale_JCVM582F2BACCAACATATGTCATGTGTACGGT
Yale_JCVM582F2AATCAGCATATGTCATGCGTACGAT
Yale_JCVM1367R2BTGTGTACTATCAATCCAGTAACATCTTCA
Yale_JCVM1367R2ATATGAACCATTAACCCAGTAATGTCTTCA
Yale_JCVM1910F4BTGGGATTTTGCAAATGAAATGAAGACA
Yale_JCVM1910F4ATGGGATTTCGCAAATGAAATGAAAACT
Yale_JCVM2747R4BTACTGCATCTGGGACTAAGGCA
Yale_JCVM2747R4ATGTTGCACCTAGGGCTTAGACA
Yale_JCVM3303F6BTCTTCGGGTCCTGTCAAGACATTA
Yale_JCVM3299F6ATGTGTATTTGGATCCTGTCAAGATATCATA
Yale_JCVM4134R6BTGGTCTACTGGTGCAAGCTCTAG
Yale_JCVM4134R6ATGGTCAACTGGTGCAAGTTCTAA
Yale_JCVL746F2BCATATGAATCAGAAAGATGGAACACTAACC
Yale_JCVL746F2ACATACGAATCAGAGAGGTGGAACA
Yale_JCVL1577R2BCATTGCCAGAATTTGGTCTCAAAAATAG
Yale_JCVL1577R2ACACTGCCAGAACCTAGTCTCAAA
Yale_JCVL1680F4BTGCATTAGTTTACCCTTCAGCTGAT
Yale_JCVL1680F4ATGCACTAGTCTACCCATCTGCA
Yale_JCVL2453R4BAGGATTTGTAAATTAACTGTTTGCTTGGT
Yale_JCVL2453R4AAGGATCTGCAAATTGACAGTTTGTTT
Yale_JCVL2940F6BTGTAGGAGAATATGAAGCCAAAATGTGC
Yale_JCVL2940F6ATGTGGGAGAATACGAGGCTAAAATG
Yale_JCVL3744R6TGTCTTTTTCATATTGGCTTGACATCC
Yale_JCVL4301F8BTGGGTGAGACAAGTGATATGAGGG
Yale_JCVL4301F8ATGGGTGAAACAAGCGATATGAGAG
Yale_JCVL5127R8BTCTTGTCATTTCTTTCAATTCAAACACAAT
Yale_JCVL5126R8ACTCGTCATCTCCTTCAGTTCAAAAAC
Yale_JCVL5681F10BTTGGTGAAGACAACAAGCTAACTTATTC
Yale_JCVL5681F10ATCGGTGAAGACAATAAGCTAACCTAC
Yale_JCVL6499R10BTAGCCTCTAAGCAACCTAAGTTTTCA
Yale_JCVL6499R10ATGGCTTCTAAACAACCTAAATTTTCTGG
Primers developed using PrimalScheme, in collaboration with New York State Wadsworth Center

cDNA & Amplification
Combine the reagents from the table below to make PCR Master Mix. Multiply each volume by the number of samples (reagent overage already included) and create separate master mixes for each primer pool (AB1 and AB2)
Reagent NameVolume per Sample
IPM15 µl
FSM3.2 µl
20 µM primer pool1.2 µl
Nuclease Free Water3.6 µl
RVT1.0 µl
Add 20 µl of PCR Master Mix to each well of a 96-well plate.
Add 5 µl of extracted RNA to each well of the 96-well plate (for a total volume of 25 µl per well)
Seal and shake at 1600 rpm for 1 minute.
Centrifuge at 280 × g for 1 minute.
Place on thermocycler and run the PCR program to generate cDNA and amplify. The thermocycler conditions can be found below

TemperatureTimeCycles
25 ºC5 minutes
50 ºC10 minutes
80 ºC5 minutes
98 ºC3 minutes
98 ºC15 seconds35 cycles
63 ºC5 minutes(included in above 35)
4 ºChold
Hybrid "one-step" cDNA generation and amplification thermocycler conditions

This is a safe stopping point, marking the end of day 1 of the protocol. Store all samples at 4 ºC
Tagment Amplicons and Cleanup
Using the Qubit, quantify the last column of samples (including both controls) from both amplicon sets.
Prepare the tagmentation master mix:
Reagent NameVolume per Sample
TB112 µl
EBLTS4 µl
Nuclease Free Water20 µl

Add 30 µl of the tagmentation master mix to 96 new PCR tubes.
Keep these new tubes on a cold block.
Combine 10 µl of the generation amplicons (pool 1 and 2) into the new tubes.
Open tubes strip-by-strip and only keep tubes you are immediately using open.
Mix for 1 minute at 1400 rpm on the plate mixer (set to 4 ºC) and spin down.
Load into the thermocycler and run the following program.
TemperatureTime
55 ºC3 minutes
10 ºChold

Once the program has finished, immediately remove tubes, spin down, and keep on a cold block.
Add 10 µl ST2 to each tube with a multichannel pipette and mix for 1 minute on the plate mixer.
Incubate at RT for 5 minutes and spin down.
Place on magnetic stand and wait until liquid is clear (a few minutes).
Remove and discard all supernatant from each well with a multichannel pipette.
Remove from the magnetic stand and add 100 µl TWB to each tube.
Mix for 1 minute at 1400 rpm on the plate mixer and spin down. Be careful not to introduce bubbles.
Repeat steps 27-29, but do not remove the second wash.
Amplify Tagmented Ampicons
Prepare the following amplification master mix:
Reagent NameVolume per Sample
EPM24
Nuclease Free Water24

Place the tubes with tagmented amplicons on the magnetic stand and wait until the liquid is clear (a few minutes).
Working is sets of 3 strips at a time, remove all TWB from each tube.
Remove any residual TWB from tubes with lower volume pipette.
Remove the tubes from the magnetic stand and add 40 µl of master mix to each tube.
Clean the foil of the index adapter plate with DNA-Away and 70% ethanol.
Add 10 µl index adapters to each tube using a multichannel pipette.
Make note of which index adapter set was used.
Mix for 1 minute at 1400 rpm on the plate mixer and spin down.
Load into the thermocycler and run the following program:
TemperatureTimeCycles
72 ºC3 minutes
98 ºC3 minutes
98 ºC20 seconds7 cycles
60 ºC30 seconds(included in above 7)
72 ºC1 minute(included in above 7)
72 ºC3 minutes
10 ºChold


Pool and Clean Up
Spin down tubes, place on magnetic stand, and wait until liquid is clear.
Transfer 5 µl of each library to new tubes.
Strips PooledVolume per Sample
1-315 µl
4-510 µl
6-125 µl

Change tips in between tubes; vortex and spin down when finished.
Transfer 55 µl (total volume - 5 µl) from each tube to a new 1.5 mL tube
Add 0.9 x total pooled volume µl to the tube and vortex.
Incubate at RT for 5 minutes
Spin down and place on magnetic stand and wait until liquid is clear.
Remove and discard supernatant; do not remove tube from magnetic rack.
Add 1000 µl of freshly prepared 80% ethanol and incubate for 30 seconds.
Repeat steps 45-46.
Remove the ethanol wash.
Add RSB and vortex to mix. Consult the following table for how much RSB to add:
Strips PooledVolume per Sample
1-230 µl
3-440 µl
5-1255 µl

Incubate at RT for 2 minutes.
Transfer total volume - 5 µl of the pooled libraries to a new 1.5 mL tube.
This is the final pooled library tube.
Quantify the library using the Qubit.
Analyze the fragment distribution using the bioanalyzer.
Protocol references
JCV ecology and evolution. forthcoming.
Bioinformatics pipeline: https://github.com/grubaughlab/DENV_pipeline