May 22, 2026
AphidMASH (Aphid Multi-Allele Scanning Haplotags)
- Virgile Ballandras1,
- Louise McNamara2,
- James Carolan3,
- Stephen Byrne2
- 1Teagasc, Crops Science Department;
- 2Teagasc;
- 3Maynooth University
Protocol Citation: Virgile Ballandras, Louise McNamara, James Carolan, Stephen Byrne 2026. AphidMASH (Aphid Multi-Allele Scanning Haplotags). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5yo34l1b/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: March 11, 2026
Last Modified: May 22, 2026
Protocol Integer ID: 313053
Keywords: Sitobion avenae, multiplex PCR, Kdr, BYDV, barley yellow dwarf viruses in irish spring, detection of yellow dwarf virus, throughput sequencing of cereal, yellow dwarf virus, aphid target, potato genomic, design of aphid target, aphid, winter barley crop, aphidmash, design of virus target, sequencing data, virus target, identification of the l1014f mutation, sequencing approach, throughput sequencing, using sequence data, genotyping method, l1014f mutation, clonal lineage, barley, irish journal of agricultural, genome, harbouring additional mutation
Funders Acknowledgements:
Teagasc Walsh Scholarship
Grant ID: 1365
Disclaimer
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Abstract
This protocol describes a multiplex-PCR amplicon sequencing approach designed to facilitate the genotyping of Sitobion avenae, the identification of the L1014F mutation, the screening of regions harbouring additional mutations known to infer insecticide resistance in other species, and the detection of yellow dwarf virus in aphids. The design of aphid targets was facilitated by re-sequencing data from 12 S. avenae clonal lineages (BioProject: PRJNA1039626 ) and the design of virus targets using sequence data from a HTS Survey (1).
This methodology is based on the original GT-seq protocol described by Campbell (2) and adapted from methods described in https://dx.doi.org/10.17504/protocols.io.e6nvw53zdvmk/v2 and (3).
1. S. Byrne, M. Schughart and V. Ballandras et al. The first survey using high-throughput sequencing of cereal and barley yellow dwarf viruses in Irish spring and winter barley crops. Irish Journal of Agricultural and Food Research. 2024. Vol. 63(1):1-16. DOI: 10.15212/ijafr-2023-0110
2. Campbell, N.R., Harmon, S.A. and Narum, S.R. (2015), Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing. Mol Ecol Resour, 15: 855-867. https://doi.org/10.1111/1755-0998.12357
3. Leyva-Pérez, M.d.l.O.; Vexler, L.; Byrne, S.; Clot, C.R.; Meade, F.; Griffin, D.; Ruttink, T.; Kang, J.; Milbourne, D. PotatoMASH—A Low Cost, Genome-Scanning Marker System for Use in Potato Genomics and Genetics Applications. Agronomy 2022, 12, 2461. https://doi.org/10.3390/agronomy12102461
Materials
Protocol materials
GelRed® Nucleic Acid Stain 10000X WaterMerck Millipore (EMD Millipore)Catalog #SCT123
Nucleic acid extraction
2h 12m 30s
Prepare the aphids by removing excess ethanol. This can be achieved by removing the aphids from the sample and placing them on absorbent tissue to air-dry for 15 seconds.
Position each aphid in an individual well of a new 96 well plate pre-filled with molecular-grade water for a few seconds to wash off any remaining ethanol, and then place each aphid back on an absorbent tissue for 00:00:30 to dry.
30s
Insert each aphid into an individual tube of the Lysing matrix Z 96 tube rack (be aware of static).
10m
Add 300 µL Quick-RNA lysing buffer from the Quick-RNA 96 kit (Zymo) into each tube and seal the tubes with lids provided and further cover with an aluminium foil sheet.
Note
Lids are fragile and can easily detach from each other, make sure that each tube is securely sealed to avoid leaks of the lysing buffer during agitation.
10m
Place the rack(s) in the the arms of a MM400 mixer mill and set the agitation for 00:30:00 at 24 Hz.
35m
After removing the rack(s) from the mixer mill, centrifuge 1000 x g, 00:02:00 and place the plate on ice.
5m
Take 200 µL of the lysate supernatant from each tube and transfer to the corresponding well of a RNAse free 96 deep-well plate. Add 200 µL of 99% ethanol to each well. Mix well by pipetting up and down.
5m
Transfer the mixture to the corresponding wells of a Silicon A‱ Plate, mounted on a Collection Plate and centrifuge 2500 x g, 18-25°C, 00:05:00 . Discard the flow-through and replace the Silicon A plate on the Collection plate.
10m
Add 400 µL / well of RNA Prep Buffer to the plate and centrifuge 2500 x g, 18-25°C, 00:05:00 . Discard the flow-through and replace the Silicon A plate on the Collection plate.
Note
The manufacturer's protocol includes an optional DNase I treatment prior to this step. This has not been used in this protocol.
10m
Add 500 µL / well of RNA Wash Buffer to the plate and centrifuge 2500 x g, 18-25°C, 00:05:00 . Discard the flow-through and repeat this step.
10m
Mount the Silicon A plate onto an Elution Plate and add 25 µL DNase/RNase-Free Water directly to the matrix and centrifuge 2500 x g, 18-25°C, 00:05:00 .
10m
The RNA can then be stored at -70 °C or you can proceed directly to cDNA synthesis.
cDNA Synthesis
1h 35m
Thaw the RNA on ice and briefly centrifuge the plate to collect contents in bottom of wells. Transfer 2 µL from each well into a new nuclease free 96 well plate. Add 1 µL of Random Hexamer Primer (Thermo Fisher Scientific, Cat: SO142), and 9.5 µL of double distilled water (ddH2O). Seal the plate with using a foil adhesive sheet and incubate the plate at 65 °C for 5 minutes .
5m
Add 4 µL of RevertAid reaction buffer , 0.5 µL of RiboLock RNase, 2 µL of dNTP Mix (10 millimolar (mM) each) and 1 µL of RevertAid reverse transcriptase (Thermo Fisher Scientific, Waltham, Massachusetts, USA; Cat: K1691) to each well for a final reaction volume of 20 µL . Seal the plate with using a foil adhesive sheet.
3m
Briefly centrifuge the plate to collect contents in bottom of wells, and place the plate into a thermal cycler and incubate for 00:10:00 at 25 °C , followed by an incubation at 42 °C for 01:00:00 and 70 °C for 00:10:00
1h 22m
The cDNA plates can then be stored at -20 °C for short term storage or -70 °C for longer term storage.
PCR One
7m 30s
PCR1 is performed with the cDNA from above as a template. This first PCR is designed to amplify the targets in the aphid and yellow dwarf virus.
PCR1 primers:
| A | B | C | |
| Target ID | Forward primer (5’to 3’) | Reverse primer (5’to 3’) | |
| AM_01 | GGGACTGTTTACACGTTGGAGA | TCAGCCGTAGGCACTGATAAA | |
| AM_02 | GGGACTGTTTACACGTTGGAGA | GCCCTCCTAAATTCTTGCACTG | |
| AM_03 | CTTCGAGTATTTAAGTTGGCAAAATC | TTTCCAAATAACTGCATACCCATAAC | |
| AM_04 | CACCACCACCACCACTTC | GGTCACCTCTGCTACGCC | |
| AM_05 | GCGATCACGTCCAACATGTAG | GCGAAGGCGTCATCAATAATG | |
| AM_06 | CGGATCGCCACTAATCAAGAG | GTGGTCGACGTGGTCATTGT | |
| AM_07 | GCATTAGACACATACCGGAAAATTC | ACAATTGGACTCCGTATAAAGTTGA | |
| AM_08 | GAAGCAAGCATGAATCTAACAAGTG | GTTAGTCTCTGGTGTTCTCGC | |
| AM_09 | GTGGCTGTTTTGGATACTTTGC | TTTCCTTCACCGATGTAGCCG | |
| AM_10 | CATTGAGACTCAGGTTGATTCAAGC | TTGAAATTAGGGTGACTGATAGAAGC | |
| AM_11 | GTAACGGCTGTTCTGACTGG | CAAAGACAGTTTGGGTTTACCG | |
| AM_12 | TGCCATCTCATCTTCCCTTTGTT | GTATGGGATAAGAAAATGGCCAAAG | |
| AM_13 | ACGAAATGTCTTGGAATAGCCTTAG | GTGGGTTGGCATGCAAAGTG | |
| AM_16 | CAGCAGCACTTCTGAATGGC | CCTTGCAGGCCAAGTTGTG | |
| AM_18 | GCGCCTCAATCCATCACAGA | CAGGGCACTTCCAATGTTCTC | |
| AM_19 | CGTGACCATTATTTGACGTGTAC | CCGAAATGTAACTGTGACGGTG | |
| AM_21 | CTCCCCACCAATCAGGACTAC | GGATTCATTTGATAGCCCATCTTG | |
| AM_22 | GCATTTTGATCGGTTCTTTGTCGA | GATAACGAATCCCAAAGCTGGAC | |
| AM_23 | GGACTAACAGCAAGTGAAGGC | GCCCTACATCGCCATCTTATAG | |
| AM_24 | CAGGCAGACGCACAACTCAG | AGCTGATACCGGGCCAGAAA | |
| AM_25 | GGTTGGAGAAGGAAACGGGG | AGTTGACGGTGGTTGTGACG | |
| AM_26 | TCGGGTTGCTGTTCCTCGT | ATGATGATCGACCTGCGCC | |
| AM_27 | TTGAGCGGAAATCACGGGAC | GCCAACACTGGTCACAGAC | |
| AM_28 | CCTGTACTCTTCCTTCAACCTTTG | CGATGAGGCGCACAATATTGAC | |
| AM_29 | TCTTGATGACGTTCATCCCGTG | AGATCTGAGTATACTGATGACGTTTC | |
| AM_30 | GGAGTTGAACAAACAGTTGCTC | AAATCTCCATGGGAGGCAGC | |
| AM_31 | AAAGAACCCGTGGTCGTGC | CTGCACGTCGTTTCGTTGAAG | |
| AM_32 | CCACCAGAACCACCTTCAATAGA | CCGGTGGAGGTGACAGTAGTT | |
| AM_33 | ATGTTGTTGTTGCAGGCCG | TGTTTCTCACGGTTCCCGAC | |
| AM_34 | GACATGCTGCACTGTGTTTGG | GTGGAGACCTGGTATAGATTGC | |
| AM_35 | GAGGTAGTCGTTGATGTCGC | GTTCGGGTGGCAGAACTTTC | |
| AM_36 | CAGATGAATACGACAAAGTGTTTGG | GCTGACACCTCAAGATCACAC | |
| AM_37 | CAGCAATTGTAGCCAATAAACGG | GGCTGTCATTTCCATCTTCAATTTG | |
| AM_38 | GGTACCGATAACGTTAGCCAC | GGAAATCCCATCACGTGACAC | |
| AM_39 | TGAACGCATGCACAAACAGT | CGCTAGTAGTTTCTCTGAGTGC | |
| AM_41 | AATTTGGATTCCACATTGTAGCACC | TAGGTTTAGGCATCCTCGCC | |
| AM_43 | GAGGTTCGGGTAATTGGTGTG | CTACATAGACTGCCTGTGCCTG | |
| AM_44 | TAGACACTCGCGTACGTACC | GCTGATATTCCCGCCACTGT | |
| AM_45 | GAGCTTTGCACATGGTGTTGAT | CAGAAACAGGGACATCATCTGC | |
| AM_46 | TTACGACGCTGTTTGCCG | AACCGTCGCTGAATCACC | |
| AM_47 | TCTAACGGTTGACAGGAGGAC | CGGTAAATGACTTGTTGCACAGG | |
| AM_48 | TATGCCCGTCACGTACAGATC | AAGTGGTACGTGAGCCGC | |
| AM_51 | GGATCCCCCACTGTGCTTTC | CCCGTGTTCCCACTTAAGCG | |
| AM_52 | AGGTGAGAGGCCACAGAATG | GCTTCTCATTGGCTCTGTCTGT | |
| AM_53 | CGACGTCAAAAGTTACATGTCGG | CATCTGTTCCATACGAAATGACG | |
| AM_54 | CAAGAGGAGGAGGGGCAAAT | CTTGAGTATTCCACCTGAAAGCG | |
| AM_55 | AATTGGCTGGTTCTGGGAGAC | GCCACCTCCGCTCTCATTT | |
| AM_56 | CTCTATTAGGACTGCCGGAGAT | AACACTCTTCTCTCCACTGCAAC | |
| AM_58 | GCATCTCTGTCAGCAAACTTGG | CCCGTGTTCCCACTTAAGCG | |
| AM_59 | GTGCGAGCGGCCAAGAAATA | GCTTCTCATTGGCTCTGTCTGT | |
| AM_61 | GGAGACGACTGTGTCATCATCA | CATTAATAGAGACCGGTTTTGACTG | |
| AM_62 | CAATCGAGCAGGACCCAGAC | GCATTGCGATAGACTGGGGC | |
| AM_63 | ATTGACTTCCTTGAAGCCATCAA | GTAGGCGGTCAAAGGTAAGC | |
| AM_64 | GGATTATCCACCGAGGACCAAG | TCCAATCGGAGACTGACCAGT | |
| AM_65 | CAGCCAGTGGTTGTGGTC | GATACTTCCAGAGGAGTTGCC | |
| AM_66 | TCTTACAGTTCGTCAGCGAGG | ATCATCTTCGCTGGGAAGCT | |
| AM_67 | TCGACAGTTCCTCACGATTCTT | GCAACGACCGAGCAGACAA | |
| AM_68 | AGCACAACCTTAGGGAAGCG | GACGCTTCCGCATCCGAA | |
| AM_69 | GCTGTCTGGTTGCAAGGCAT | GCCACAATGATAGGCAGCCA | |
| AM_70 | GCAGTTCCGATGTTTGTCTTTCC | AGCCCCAATATTCCACCCTTC | |
| AM_71 | GCTGACAACAGTTGGAAGTACG | GAAGTTTTCTGGACAGGCGGA |
PCR 1 primers pairs designed to target aphid’s and viruses' genomes. These primers were retained after verifying that they targeted unique regions in the genome and that no primer–primer interactions would occur during PCR.
The targets AM_01 AM_02 and AM_03 were designed to amplify the regions of the
VGSC including kdr and super-kdr mutations.
The targets AM_04 to AM_48 were designed to amplify regions of the aphids' genome to analyse their genetic diversity.
The targets AM_51 to AM_62 were designed to amplify regions of BYDV's genome.
The targets AM_63 to AM_71 were designed to amplify regions of BYDV's genome.
Note
All PCR1 primers are composed of the sequence matching to the organism’s genome (aphid or virus) and Illumina sequencing primer tags attached to the 5’ end of the forward primer and the 5’ end of the reverse complement of the reverse primer.
These sequences, R1: 5’ CACCACCACCACCACTTC 3’ and R2: 5’ CCATGGAGACGATGCGG 3’,
for the forward primer and the reverse primer respectively, served as anchorage regions for the PCR2 primers.
Prepare the primer mix dilution:
Put one volume of each primer (forward and reverse), delivered at concentration 500 micromolar (µM) , into a tube and complete the dilution with three volumes of ddH2O and mix thoroughly by pipetting.
Note
This brings the primer mix's concentration to 4 micromolar (µM) for each primer.
Take a fraction of this solution and dilute it into three volume of ddH2O equivalent to the volume of this fraction to bring the final concentration of the primer mix to 1 micromolar (µM) , for each primer.
Reaction Mix preparation in each of the 96 well:
12.5 µL Multiplex PCR master Mix
2 µL of the primer pool solution at 1 micromolar (µM) , each primer
9.5 µL of ddH2O
1 µL of template cDNA to the respective well
Note
Tip:
Prepare a large volume of reaction mix in a tube (corresponding to 100 - 104 wells), without the template cDNA. Distribute the reaction mix in the wells using a multichannel pipette.
Add the template cDNA using the multichannel pipette afterward. this saves time and reduce the risks of cross-contamination.
Note
If out of the freezer from long time storage, taw the cDNA plate on ice in the lab and briefly centrifugate the plate in a bench plate centrifuge if available.
Seal the plate with an aluminium sticker. Briefly centrifugate the plate in the bench bench plate centrifuge.
Thermo-Cycling:
Place the PCR plate in a
Equipment
MiniAmp Plus Thermal Cycler
NAME
Applied Biosystems
BRAND
A37835
SKU
and run the following PCR cycle:
95 °C 00:05:00
followed 30 cycles of :
95 °C 00:00:30
a ramp down temperature decrease 2 °C / second until reaching 60 °C
72 °C 00:02:00
At the end of the cycles, keep the plate at 10 °C . Remove the PCR plate from the thermal cycler, let it cool down on ice and spin it in the bench bench plate centrifuge for one or two minutes.
Note
At this stage, the PCR 1 product can be stored at -70 °C for long time storage, -20 °C for short time storage or directly used for PCR2
Note
Cross contamination can occur until the end of PCR2, do not pool or mix the well's content before the end of PCR2. Opening the plate sealing without centrifugating or too abruptly can lead to cross contamination.
If cross-contamination is induced during PCR1, restart PCR1, do not proceed to PCR2 with a low quality PCR1
PCR 1 quality can be assessed with a
Equipment
Nanodrop 2000C
NAME
Thermo Scientific
BRAND
TSC-ND2000C
SKU
and / or a migration of the PCR product in an agarose gel, expecting an amplicon's length of about 150 bp
7m 30s
PCR Two
Prepare a dilution of the PCR1 product by pipetting one volume of the PCR1 product into the respective wells of a new nuclease free 96 well plate. Add 14 volumes of ddH2O to each well. Seal with an aluminium sticker after mixing by pipetting.
PCR2 primers:
PCR2 primers are composed of an Illumina sequencing adaptor, a 6nt barcode used to index the position of each well into each plate, the sequencing primer tag:
Reverse primer I7 indexes:
| A | B | |
| Plate ID | Index sequence 5’-3' | |
| Plate 1 | ATCACG | |
| Plate 2 | CGATGT | |
| Plate 3 | TTAGGC | |
| Plate 4 | TGACCA | |
| Plate 5 | ACAGTG | |
| Plate 6 | GCCAAT |
Forward primer I5 indexes plate layout:
| A | |
| A1_ATCACG | |
| B1_CGATGT | |
| C1_TTAGGC | |
| D1_TGACCA | |
| E1_ACAGTG | |
| F1_GCCAAT | |
| G1_CAGATC | |
| H1_ACTTGA | |
| A2_GATCAG | |
| B2_TAGCTT | |
| C2_GGCTAC | |
| D2_CTTGTA | |
| E2_AGTCAA | |
| F2_AGTTCC | |
| G2_ATGTCA | |
| H2_CCGTCC | |
| A3_GTAGAG | |
| B3_GTCCGC | |
| C3_GTGAAA | |
| D3_GTGGCC | |
| E3_GTTTCG | |
| F3_CGTACG | |
| G3_GAGTGG | |
| H3_GGTAGC | |
| A4_ACTGAT | |
| B4_ATGAGC | |
| C4_ATTCCT | |
| D4_CAAAAG | |
| E4_CAACTA | |
| F4_CACCGG | |
| G4_CACGAT | |
| H4_CACTCA | |
| A5_CAGGCG | |
| B5_CATGGC | |
| C5_CATTTT | |
| D5_CCAACA | |
| E5_CGGAAT | |
| F5_CTAGCT | |
| G5_CTATAC | |
| H5_CTCAGA | |
| A6_GACGAC | |
| B6_TAATCG | |
| C6_TACAGC | |
| D6_TATAAT | |
| E6_TCATTC | |
| F6_TCCCGA | |
| G6_TCGAAG | |
| H6_TCGGCA | |
| A7_AAGTAC | |
| B7_TGCCAT | |
| C7_CTCCTT | |
| D7_AGATAG | |
| E7_GATATA | |
| F7_ATAATA | |
| G7_GGTGCT | |
| H7_TGTGGA | |
| A8_CGAGAA | |
| B8_GCAGGA | |
| C8_GCCGCG | |
| D8_AGCGCA | |
| E8_TCCGTT | |
| F8_ATCGTC | |
| G8_CCGCAA | |
| H8_AACTTG | |
| A9_AAGGTA | |
| B9_GCGCTC | |
| C9_CCTCGT | |
| D9_ACGATT | |
| E9_AGACTC | |
| F9_TGGTCT | |
| G9_GGAATT | |
| H9_ACATCT | |
| A10_TTCTCC | |
| B10_ATCTAT | |
| C10_TGCATA | |
| D10_AATAGG | |
| E10_CGGTTA | |
| F10_GAGACT | |
| G10_GCTCCA | |
| H10_ATGCAG | |
| A11_GGCCTG | |
| B11_TTACTG | |
| C11_TCAAGT | |
| D11_AGCAGT | |
| E11_CCTTAG | |
| F11_TTGGAT | |
| G11_TCTCAC | |
| H11_GTCTTA | |
| A12_CTAACG | |
| B12_ATACAC | |
| C12_GCATTC | |
| D12_TACTCT | |
| E12_TGGACT | |
| F12_CCATAG | |
| G12_TAAGGT | |
| H12_TTGGCG |
Prepare a dilution of the PCR2 primers to 10 micromolar (µM) :
PCR2 primers were delivered at concentration 100 micromolar (µM) .
In a new nuclease-free 96 well plate, mix 1 volume of each primer (forward) and 9 volume of ddH2O in the respective primer's well.
In a new nuclease-free tube, mix 1 volume of one of the reverse primer and 9 volume of ddH2O.
Note
Do not mix PCR 2 primers that have different I5 or I7 indexes
PCR mix in each well of a new nuclease free 96 well PCR plate for a final volume of 25 µL per well:
12 µL of Multiplex PCR Master Mix
8 µL of ddH2O
1 µL of Forward primer 10 micromolar (µM)
1 µL of Reverse primer 10 micromolar (µM)
3 µL of the diluted PCR1 product
Thermo-Cycling:
Place the PCR plate in a
Equipment
MiniAmp Plus Thermal Cycler
NAME
Applied Biosystems
BRAND
A37835
SKU
and run the following PCR cycle:
95 °C for 00:15:00
10 cycles of:
98 °C for 00:00:10
65 °C for 00:00:30
72 °C for 00:00:30
at the end of the cycles:
hold at 72 °C for 00:05:00 and keep at 10 °C
Remove the plate from the thermal cycler and centrifugate in the bench plate centrifuge and keep on ice if used directly for the next steps.
PCR2 products can be stored at -20 °C for short time storage and -80 °C for long time storage.
Preparation of Libraries for Sequencing
PCR2 product normalisation:
This step is performed using the NGS Normalization 96-Well Kit:
Prepare a working concentration of Wash Solution A by adding 90 mL of 96 - 100% ethanol
Add 75 µL of Buffer SK directly to each well of the PCR2 plate, Mix by pipetting up and down and centrifuge the plate.
Place the 96-Well Normalization Plate on top of a provided 96-Well Collection Plate and add the mixture from the PCR2 plate into the wells of the 96-Well Normalization Plate.
Centrifuge the assembly at maximum speed or 3200 x g, 00:02:00 .
Discard the flowthrough. Reassemble the 96-Well Normalization Plate and the 96-Well Collection Plate
Note
Ensure that all of the lysate from each well has passed through into the bottom plate. If the entire lysate volume has not passed, centrifuge for an additional 2 minutes.
Apply 400 µL of Wash Solution A to each well of the 96-Well Normalization Plate.
Centrifuge the assembly at maximum speed or 3200 x g, 00:02:00 .
Note
Ensure that all of the lysate from each well has passed through into the bottom plate. If the entire lysate volume has not passed, centrifuge for an additional 2 minutes.
Discard the flowthrough. Reassemble the 96-Well Normalization Plate and the 96-Well
Collection Plate
Repeat step 26.4 for a second wash and Centrifuge the assembly at maximum speed or 3200 x g, 00:15:00 .
Gently pat the bottom of the 96-Well Normalization Plate on paper towels.
Stack the 96-Well Normalization Plate and the 96-Well Elution Plate (provided).
Add 100 µL of Elution Buffer B to each well of the 96-Well Plate.
Centrifuge the assembly at 200 x g, 00:01:00 followed by 3200 x g, 00:04:00
Pooling libraries, Concentration - Purification:
At this stage,PCR2 products are barcoded and normalized.
Pool the normalized products in a single tube.
The PCR product purification is performed using the QIAquick PCR Purification Kit and a bench centrifuge.
Before starting, add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
Sub-sample a volume of the PCR2 pooled product mix and add 5 volumes of Buffer PB into a new nuclease-free falcon tube.
Prepare collection tubes with a QIAquick Spin column inserted in each tube.
Note
Ideally, prepare 4 of them as important volumes of libraries will be purified. While 2 tube-column assemblies will be spinning, the two other ones can be filled with the solution to purify.
This will be repeated multiple times, until the totality of mix of PCR2 product with Buffer PB passed through.
Processing one library (corresponding to one 96 well plate) at the time is recommended. in this case, it is not necessary to label the columns or the collection tubes as they contain the same solution.
If multiple libraries are processed at the same time, always label correctly all the tubes and columns to not mix the libraries.
Proceed to the filtering of the mix of PCR2 product with Buffer PB to bind DNA by applying the mix to the QIAquick column and centrifuge 17900 x g, 00:01:00 .
Repeat as many times as necessary with the same set of columns to filter all the mix. Discard the flowthrough each time and re-incert the QIAquick Spin column in its collection tube.
Note
Do not put more than 600 - 650 µL at the time otherwise the flowthrough will touch the bottom of the column.
To wash, add 700 µL of Buffer PE to the QIAquick column and centrifuge 17900 x g, 00:01:00 .
Discard flow-through and place the QIAquick column back into the same tube. Centrifuge 17900 x g, 00:01:00 . The residual ethanol from the Buffer PE must be dried.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µL of Buffer EB (10 mM Tris·Cl, pH 8.5) or nuclease free water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 17900 x g, 00:02:00 .
Size selection:
As mentioned in the PotatoMASH library construction protocol:
"This step is meant to remove PCR1 primer-dimer amplified products in PCR2 and other unwanted secondary products from the libraries. All core primers are designed to produce libraries of around 300bp. Therefore, electrophoresis in agarose gel is a low-cost and easy way to separate them."
Make a 2% agarose gel and pour the totality of the purified library from step 27.5 in the "wide wells" formed in the gel.
Note
"Wide wells can be achieved, for example, by covering three 7 mm. wide x 1mm thick comb teeth (3 wells) with adhesive tape to make a well of 2.5 cm x 1mm thick."
Elution Buffer EB from QIAquick PCR Purification Kit tend to have a lower density than the TBE buffer in which the agarose gel is plunged for the electrophoresis. To avoid loosing the purified library in the buffer it is highly recommended to increase the volume of gel loading buffer compared to a standard electrophoresis.
Before to load the libraries in the gel, prepare loading mix composed of 70% Purified library and 30% loading buffer. Mix thoroughly by pipetting, centrifuge in a bench centrifuge for 00:00:30 and load the mix into the wide wells.
Perform the migration at 85 V during 03:00:00 to clearly separate DNA fragments from different sizes.
Remove the gel from the migration tank and simmer in TBE buffer containing some GelRed® Nucleic Acid Stain 10000X WaterMerck Millipore (EMD Millipore)Catalog #SCT123 , placed on an orbital shaker, agitation about 40 - 45 rpm, 01:00:00
Place the gel on a transilluminator UV (recommended to use low intensity like 70%) to visualise the DNA bands and excise slices between 270 and 300 bp using a scalpel and place each slice in a 2 mL tube.
Note
Gel slices can be stored overnight at low temperature 4 °C (DO NOT freeze the gel slices).
However it is recomanded to perform the extraction of the DNA from the gel slices strait after the exicion.
Gel extraction and clean up:
This step is performed using the Wizard‱ SV Gel and PCR Clean-Up System
Weigh each gel slice and add 10 µL of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is completely dissolved.
Insert SV Minicolumn into Collection Tube and Transfer dissolved gel mixture to the Minicolumn assembly. Incubate Room temperature 00:01:00 .
Centrifuge 16000 x g, 00:01:00 for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube
Add 700 µL of Membrane Wash Solution (ethanol added). Centrifuge at 16000 x g, 00:01:00 . Discard flowthrough and reinsert Minicolumn into the collection Tube.
Repeat this with 500 µL of Membrane Wash Solution. Centrifuge at 16000 x g, 00:05:00 .
Empty the Collection Tube and recentrifuge the column assembly 16000 x g, 00:01:00 with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol
Carefully transfer Minicolumn to a new 1.5ml microcentrifuge tube and add 50 µL of Nuclease-Free Water to the Minicolumn.
Incubate Room temperature 00:01:00 .
Centrifuge at 16000 x g, 00:01:00 .
Discard Minicolumn and store DNA at 4 °C for very short storage (ex. overnight) or -20 °C for a few days.
Last PCR purification using Agencourt AMPure XP magnetic beads:
Mix 1.8 volumes of the AMPure XP magnetic bead solution with 1 volume of purified library into a new nuclease free 1.5 mL tubes. Mix thoroughly by pipetting carefully up and down about 20 times.
Be careful not to pipet too fast or abruptly to avoid damaging the DNA fragments (the longest the fragments, the more fragile they are).
Incubate Room temperature 00:05:00 .
Place the tube on a magnetic rack, compatible with 1.5 mL tubes and wait for the beads to bind to the wall of the tube (magnet side).
The solution must be clear before to perform the next steps.
Remove the cleared solution by pipetting. Do not disturb the beads aggregate or aspirate some beads.
If beads are pipetted up, put the solution back in the tube, wait for the solution to clear again and restart pipetting.
The tube stays on the magnetic rack.
Immerge the beads into 70% ethanol and incubate Room temperature 00:00:30 .
Remove the ethanol by pipetting, without disturbing the beads.
Repeat step 30.4 and let the bead aggregate to dry for a couple of minutes with the lid open.
Before the bead aggregate to dry completely and appear cracked, remove the tube from the magnetic rack and add 40 µL of ddH2O to perform the elution.
Carefully mix by pipetting up and down about 20 times the beads into the water.
incubate Room temperature 00:02:00 .
Replace the tube on the magnetic rack and wait for the beads to aggregate again on the tube's wall.
Carefully pipette up the cleared water containing the purified DNA and place it into a new nuclease free tube.
Note
Do not pipet any beads, it is preferable to leave a few microliters of water with the beads instead of including beads in the final eluted product.
The final library can now be frozen at -20 °C or -80 °C depending on the use and be sent for sequencing
Final library quality control
It is recommended to check the size of the DNA fragments included in the final library, as well as the quantity of DNA.
Make a 2% agarose gel and migrate 2 µL of the final library in the TBE agarose gel at 85 V during 02:00:00 . Proceed as in and to observe the bands corresponding to the DNA fragments.
Asses the DNA concentration of the final library using:
Equipment
Qubit fluorometer
NAME
designed to measure DNA, RNA, and protein quantity or quality using the Invitrogen Qubit RNA IQ (integrity & quality) assay.
TYPE
ThermoFisher
BRAND
Q33238
SKU
Broad range assay. Use a volume of 2 µL of the final library to be measured.