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Nov 2020
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VirScan: High-throughput Profiling of Antiviral Antibody Epitopes
Vir Scan: 抗病毒抗体表位的高通量分析   

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Abstract

Profiling the specificities of antibodies can reveal a wealth of information about humoral immune responses and the antigens they target. Here, we present a protocol for VirScan, an application of the phage immunoprecipitation sequencing (PhIP-Seq) method for profiling the specificities of human antiviral antibodies. Accompanying this protocol is a video of the experimental procedure. VirScan and, more generally, PhIP-Seq are techniques that enable high-throughput antibody profiling by combining high-throughput DNA oligo synthesis and bacteriophage display with next-generation sequencing. In the VirScan method, human sera samples are screened against a library of peptides spanning the entire human viral proteome. Bound phage are immunoprecipitated and sequenced, identifying the viral peptides recognized by the antibodies. VirScan Is a powerful tool for uncovering individual viral exposure histories, mapping the epitope landscape of viruses of interest, and studying fundamental mechanisms of viral immunity.


Graphical abstract:



Keywords: VirScan (VirScan), PhIP-Seq (PhIP-Seq), Bacteriophage display (噬菌体展示技术), Synthetic biology (合成生物学), High-throughput screening (高通量筛选), Serology (血清学), Antibody (抗体), Epitope (表位), Virus (病毒), Immunology (免疫学)

Background

VirScan (Xu et al., 2015) is based on a general technology called phage immunoprecipitation sequencing (PhIP-Seq) (Larman et al., 2011; Mohan et al., 2018 ; Mandel-Brehm et al., 2019, Garrett et al., 2020). In PhIP-Seq, a proteome-scale library of peptides is designed, and DNA oligos encoding these peptides are synthesized and cloned into a T7 bacteriophage display system. Each phage encodes the sequence of one peptide in its genome and displays the same peptide on its surface, thus linking genotype with phenotype (Smith and Petrenko, 1997; Kosuri et al., 2010). For each PhIP-Seq reaction, the phage display library is mixed with a sample containing human antibodies, and the antibodies bind to their cognate epitopes on the phage surface. Then the phage-antibody complexes are immunoprecipitated and unbound phage are washed away. PCR amplification and high-throughput sequencing of the insert DNA from bound phage reveal the peptides targeted by antibodies in the sample. Whereas the original PhIP-Seq assays were performed using a phage display library of peptides derived from the human proteome to detect autoantibodies, VirScan employs a library of peptides derived from the human virome to identify the specificities of antibodies targeting viral antigens.


A comparison of the advantages and disadvantages of PhIP-Seq relative to peptide or protein microarrays for high-throughput epitope profiling is provided in Mohan et al. (2018). In brief, PhIP-Seq enables higher throughput, less expensive, and more highly programmable assays relative to peptide and whole protein microarrays. A disadvantage of PhIP-Seq compared with protein microarrays is that the experimental procedure takes longer to perform since its readout involves next-generation sequencing. As with all peptide-based epitope profiling methods, PhIP-Seq generally does not enable detection of discontinuous epitopes or epitopes that involve post-translational modifications.


A comprehensive article describing the PhIP-Seq protocol has been published (Mohan et al., 2018 ) and is a valuable resource to those interested in using VirScan technology. The present article serves to supplement that resource by presenting videos of the experimental protocol (Videos 1–5) and including information relevant for VirScan-specific data analysis (Supplementary materials). This protocol does not contain information related to the design and generation of the VirScan phage display library, as these methods have been covered in depth by Mohan et al. (2018) and Xu et al. (2015). This article assumes that the researcher has access to the VirScan library and focuses on the downstream experimental procedures, namely, phage-antibody complex formation, immunoprecipitation, and preparation of DNA libraries for next-generation sequencing, which are also depicted in the videos (Videos 1–5). Further, this protocol covers VirScan-specific data analysis steps, including hits by virus calculation, virus score calculation, and determination of virus seropositivity.


The VirScan protocol may be modified with supplemental libraries, alternative immunoprecipitation reagents, and input samples other than human serum to address a broader set of scientific questions. Alanine scanning and saturation mutagenesis libraries may be designed to enable high-resolution mapping of antibody epitopes, as performed in Shrock et al. (2020) and Chen et al. (2021). The standard immunoprecipitation reagents, Protein A and Protein G, may be replaced with isotype-specific secondary antibodies to profile antibody isotypes other than IgG, such as IgA or IgE, as performed in Shrock et al. (2020) and Chen et al. (2021). The protocol may be used with serum samples from several mammalian species other than humans, including mice and non-human primates, since the Protein A and Protein G bind to mouse and non-human primate IgG as well as human IgG (Borriello et al., 2022). Finally, antibody-containing samples other than serum, including saliva, breast milk, and supernatant from cultured B cells, may be used as input samples for VirScan.


VirScan has been used successfully in many applications, including to estimate the number of viral species to which individuals have been exposed (Xu et al., 2015); to show that infection by measles virus diminishes the preexisting antibody repertoire, leaving individuals vulnerable to reinfection to pathogens (Mina et al., 2019); to study the maternally derived antibody repertoire in human infants (Pou et al., 2019); to investigate the effects of CART therapy directed against CD19 on the antiviral antibody repertoire (Hill et al., 2019); to uncover a putative viral etiology of the rare neurological condition Acute Flaccid Myelitis (Schubert et al., 2019), to map SARS-CoV-2 linear epitopes with high resolution and determine humoral immune correlates of COVID-19 severity (Shrock et al., 2020; Zamecnik et al., 2020), and to provide evidence that Epstein-Barr virus infection increases risk for subsequent development of multiple sclerosis (Bjornevik et al., 2022 ).

Materials and Reagents

  1. Pipette Tips SR LTS 20 µL F 960A/5 (Rainin, catalog number: 17005860), storage temperature: room temperature

  2. Pipette Tips SR LTS 200 µL F 960A/5 (Rainin, catalog number: 17005859), storage temperature: room temperature

  3. Pipette Tips SR LTS 1,200 µL F 768A/4 (Rainin, catalog number: 17007084), storage temperature: room temperature

  4. Disposable Serological Pipets, Polystyrene, Sterile, Plugged, 5 mL (e.g., VWR, catalog number: 89130-896), storage temperature: room temperature

  5. Disposable Serological Pipets, Polystyrene, Sterile, Plugged, 10 mL (e.g., VWR, catalog number: 89130-898), storage temperature: room temperature

  6. Disposable Serological Pipets, Polystyrene, Sterile, Plugged, 25 mL (e.g., VWR, catalog number: 89130-900), storage temperature: room temperature

  7. Disposable Serological Pipets, Polystyrene, Sterile, Plugged, 50 mL (e.g., VWR, catalog number: 89130-902), storage temperature: room temperature

  8. Reagent Reservoirs, Sterile (e.g., Corning, Costar, catalog number: 4870), storage temperature: room temperature

  9. Sterile Filter Storage Bottles/Receivers (e.g., Thermo Fisher, Nalgene, catalog number: 455-0500), storage temperature: room temperature

  10. Deep Well Plate, 96-well, PP, 1.1 mL, Standard, U-Bottom (Cole-Parmer, BrandTech, catalog number: EW-07904-04), storage temperature: room temperature

  11. Kimtech Science Kimwipes Delicate Task Wipes (Kimberly-Clark, catalog number: 34155), storage temperature: room temperature

  12. Sealing paddle (USA Scientific, catalog number: 2928-7355), storage temperature: room temperature

  13. MicroAmp Optical Adhesive Film (Thermo Fisher, Applied Biosystems, catalog number: 4311971), storage temperature: room temperature

  14. Colored Labeling Tape, Rainbow Pack (Fisher Scientific, Fisherbrand, catalog number: 15-901-10R), storage temperature: room temperature

  15. PCR Plate, 96-well (e.g., VWR, catalog number: 82006-704), storage temperature: room temperature

  16. Bravo Lab Disposable Pipette Tips (Agilent, catalog number: 19477-022), storage temperature: room temperature

    Note: These are necessary if performing magnetic bead washes using the Agilent Bravo.

  17. Nunc 96-Well Polypropylene DeepWell Storage Plates, sterile (Thermo Fisher, Thermo Scientific, catalog number: 260251), storage temperature: room temperature

  18. Nalgene Disposable Polypropylene Robotic Reservoirs, sterile (Thermo Fisher, Thermo Scientific, catalog number: 1200-1301), storage temperature: room temperature

    Note: These are necessary if performing magnetic bead washes using the Agilent Bravo.

  19. Corning 96-well Clear V-Bottom 2 mL Polypropylene Deep Well Plate, sterile (Corning, catalog number: 3960), storage temperature: room temperature

    Note: These are necessary if performing magnetic bead washes using the Agilent Bravo.

  20. MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL (Thermo Fisher, Applied Biosystems, catalog number: 4346906), storage temperature: room temperature

    Note: These are necessary if performing qPCR using the Applied Biosystems Fast 7500 system.

  21. Qubit Assay Tubes (Thermo Fisher, Invitrogen, catalog number: Q32856), storage temperature: room temperature

  22. (Optional) IgG (Total) Human ELISA Kit (e.g., Thermo Fisher, Invitrogen, catalog number: BMS2091), storage temperature: 4°C

  23. Tris Buffered Saline with Tween 20 (TBST-10X) (Cell Signaling, catalog number: 9997), storage temperature: room temperature

  24. Bovine Serum Albumin (BSA) (VWR, catalog number: 0332-500G), storage temperature: 4°C

  25. PBS, pH 7.4 (e.g., Thermo Fisher, catalog number: 10010023), storage temperature: room temperature

  26. VirScan T7 phage display library (Available upon request, storage temperature: -80°C)

    Note: Based on T7Select Packaging Kit (Millipore-Sigma, catalog number: 70014) storage temperature: -80°C.

  27. UltraPure 1M Tris-HCI, pH 8.0 (Thermo Fisher, Invitrogen, catalog number: 15568025), storage temperature: 4°C

  28. NaCl (5 M), RNase-free (Thermo Fisher, Invitrogen, catalog number: AM9759), storage temperature: room temperature

  29. Magnesium sulfate solution (Millipore Sigma, catalog number: M3409-100ML), storage temperature: room temperature

  30. Chloramphenicol (Millipore Sigma, catalog number: C0378-100G), storage temperature: room temperature for powder or -20°C for reconstituted solution

  31. Kanamycin B sulfate salt (Millipore Sigma, catalog number: B5264-250MG), storage temperature: -20°C for powder and for reconstituted solution

  32. NP-40 Surfact-Amps Detergent Solution (Thermo Fisher, catalog number: 85124), storage temperature: room temperature

  33. Dynabeads Protein A for Immunoprecipitation (Thermo Fisher, Invitrogen, catalog number: 10008D), storage temperature: 4°C

  34. Dynabeads Protein G for Immunoprecipitation (Thermo Fisher, Invitrogen, catalog number: 10009D), storage temperature: 4°C

  35. UltraPure 1 M Tris-HCI Buffer, pH 7.5 (Thermo Fisher, Invitrogen, catalog number: 15567027), storage temperature: 4°C

  36. UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher, Invitrogen, catalog number: 10977023), storage temperature: room temperature

  37. Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs, catalog number: M0493L) storage temperature: -20°C

  38. dNTP Set (100 mM) (Thermo Fisher, Invitrogen, catalog number: 10297018), storage temperature: -20°C

  39. TaqMan Gene Expression Master Mix (Thermo Fisher, Applied Biosystems, catalog number: 4369016), storage temperature: 4°C

  40. UltraPure Agarose (Thermo Fisher, Invitrogen, catalog number: 16500100), storage temperature: room temperature

  41. UltraPure DNA Typing Grade 50× TAE Buffer (Thermo Fisher, Invitrogen, catalog number: 24710030), storage temperature: room temperature

  42. QIAquick Gel Extraction Kit (250) (QIAGEN, catalog number: 28706), storage temperature: room temperature

  43. QIAquick PCR Purification Kit (250) (QIAGEN, catalog number: 28106), storage temperature: room temperature

  44. Qubit dsDNA HS Assay Kit (Thermo Fisher, Invitrogen, catalog number: Q32851), storage temperature: mixed, room temperature and 4°C

  45. HPLC-purified primers (IDT, storage temperature: -20°C)

    Primer name Primer sequence (5’ – 3’)
    IS7 ACACTCTTTCCCTACACGACTCCAGTCAGGTGTGATGCTC
    IS8 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCGAGCTTATCGTCGTCATCC
    IS4 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACTCCAGT
    Index primer CAAGCAGAAGACGGCATACGAGATNNNNNNNGTGACTGGAGTTCAGACGTGT
    5’NEST-qPCR TCGGGGATCCAGGAATTC
    3’NEST-qPCR CGTCGTCATCCTTGTAATCG
    NEST_probe /56-FAM/TAATCGCGG/ZEN/CCGCAAGCTTGTC/3IABkFQ/
    T7-Illumina-READ1-A TGCTCGGGGATCCAGGAATTCCGCTGCGT

    Note: Orthogonal 7 nt barcodes for the Index primer are available upon request.

  46. Phage extraction buffer (Recipe listed below), storage temperature: 4°C

  47. PhIP-Seq Wash Buffer (Recipe listed below), storage temperature: 4°C

Equipment

  1. Pipet-Lite Multi Pipette L12-20XLS+ (Rainin, catalog number: 17013808)

  2. Pipet-Lite Multi Pipette L12-200XLS+ (Rainin, catalog number: 17013810)

  3. E4 Pipette Multi E12-1200XLS+ (Rainin, catalog number: 17014499)

  4. Portable Pipet-Aid XP Pipette Controller (Drummond, catalog number: 4-000-101)

  5. Rotator (e.g., Barnstead/Thermolyne, model: 415110)

  6. Benchtop Centrifuge with swinging-bucket rotor assembly and microplate carrier (e.g., Beckman Coulter, model: Allegra X-15R or Avanti J-15R, swinging-bucket rotor assembly: SX4750A or JS-4.750 , microplate carrier: SX4750)

  7. Bravo NGS Automated Liquid Handling Platform (Agilent, catalog number: G5573AA)

  8. 96-Well Microtiter Plate Magnetic Separation Rack (NEB, catalog number: S1511S)

    Note: This is necessary if performing magnetic bead washes manually.

  9. Thermal cycler (e.g., Bio-Rad, model: C1000 Touch with 96-well Fast Reaction Module, catalog number: 1851196)

  10. 96-well Aluminum Block For 0.2 mL Tubes (Universal Medical, catalog number: 81001)

  11. 7500 Fast Dx Real-Time PCR Instrument, with laptop computer (Thermo Fisher, Applied Biosystems, catalog number: 4406984)

  12. Qubit 4 Fluorometer (Thermo Fisher, Invitrogen, catalog number: Q33238)

Software

  1. bowtie ( Langmead et al., 2009 )

  2. samtools ( Li et al., 2009 )

  3. python (Python Software Foundation. Python Language Reference, version 2.7. Available at http://www.python.org)

  4. gcc (GNU Compiler Collection, version 6.2.0. Documentation at https://gcc.gnu.org/onlinedocs/gcc.pdf)

  5. R (R Core Team, 2017)

Procedure

Video 1. Introduction


  1. Block plates (Video 2)


    Video 2. Blocking plates


    1. Prepare 100 mL of TBST 3% BSA for every 96-well deep well plate (Cole-Parmer) to be blocked. Transfer to reagent reservoir.

    2. Add 1 mL of TBST 3% BSA to each well of 96-well deep well plate.

    3. Blot top of plate with kimwipes to remove excess liquid.

    4. Seal plate well with MicroAmp optical adhesive film.

      Note: Plate may be sealed by pressing outward from the center of plate to eliminate large air bubbles, then by using a sealing paddle to eliminate all remaining air pockets.

    5. Invert plate several times to ensure the liquid is moving throughout the plate.

    6. Tape 96-well deep well plate to the plate rotator at 4°C and rotate end-over-end overnight.


  2. Phage-antibody complex formation (Video 3)


    Video 3. Phage-antibody complex formation


    1. Thaw VirScan T7 phage library on ice. Characteristics of the library are shown in Table 1.

    2. Prepare 110 mL of diluted T7 phage library for each 96-well deep well plate. See Table 2 for information on how to prepare the diluted phage library.

    3. Mix very well.


      Table 1. VirScan T7 phage library characteristics

      Complexity (version Vir3) 115,753 members
      Desired final concentration 2 × 105 pfu/mL per member of the library, or approximately 2 × 1010 pfu/mL


      Table 2. Preparing diluted phage library

      Component Stock concentration Final concentration 110 rxns
      VirScan phage library 9.9 × 1010 pfu/mL (this may vary by batch) 2 × 1010 pfu/mL 22.2 mL
      chloramphenicol 50 mg/mL (1,000×) 50 μg/mL (1×) 110 μL
      kanamycin 50 mg/mL (1,000×) 50 μg/mL (1×) 110 μL
      Phage extraction buffer To 110 mL


    4. Make aliquots of serum diluted to 0.2 μg/μL in PBS in 96-well PCR plates.

      Notes:

      1. Concentration of IgG in human serum is generally 5–10 μg/μL. Dilute 2 μL of serum in 98 μL of PBS (1:50 dilution) to reach a concentration of approximately 0.2 μg/μL human IgG. Mix well.

      2. If needed, the concentration of IgG in a sample can be measured by IgG ELISA.

      3. Serum samples are typically run in duplicate.

      4. Eight no-serum controls are typically included for each run.

    5. Pour out blocking solution from 96-well deep well plates into sink. Flick plates several times to remove all blocking solution.

    6. Blot the surface of the plate with a kimwipe to remove liquid.

    7. Add 1 mL of diluted phage library to each well.

    8. Blot the surface of the plate with a kimwipe to remove excess liquid.

    9. Add sera containing 2 μg of IgG to each well, or 10 μL of the 0.2 μg/μL plate previously prepared.

    10. Blot the surface of the plate with a kimwipe to remove excess liquid.

    11. Seal plates extremely well with a new MicroAmp optical adhesive film, using a paddle. Make sure no air bubbles remain between wells.

    12. Invert plate several times to ensure that liquid is moving throughout the plate. Secure plates on rotator at 4°C and rotate with end-over-end mixing for 20 h or overnight.

    13. Seal plate with diluted serum samples and store at -80°C.


  3. Immunoprecipitation (Video 4)


    Video 4. Immunoprecipitation


    1. Centrifuge 96-well deep well plate at 500× g for 3 min to collect liquid away from seal.

    2. Tightly hold down plate while removing seal. Avoid splashing and cross-contamination between wells.

    3. Resuspend Protein A and Protein G Dynabeads by shaking bottles until there are no remaining beads settled at the bottom.

    4. For each 96-well deep well plate, add 2 mL of Protein A and 2 mL of Protein G Dynabeads to a reagent reservoir and mix with a serological pipette.

    5. Add 40 μL of Protein A/G to each well of the 96-well deep well plate.

    6. Blot the surface of the plate with a kimwipe to remove excess liquid.

    7. Seal plate with a new MicroAmp optical adhesive seals and tape plate to a rotator. Rotate for 4 h at room temperature or overnight at 4°C.

    8. Take one plate off at a time for washes. Centrifuge the 96-well deep well plate at 500 × g for 3 min to collect liquid away from the seal.

    9. Perform three washes using a liquid handling robot, as shown in Video 4, washing with 170 μL of PhIP-Seq Wash Buffer each time. At one point during the washes, transfer beads to a new 96-well deep well plate (Thermo Fisher) to avoid phage that may have bound non-specifically to the wells of the original plate. The Bravo protocol file is available in the Supplementary materials.

      1. Alternatively, perform washes manually.

        1. Place the plate on a magnetic separation rack.

        2. Let plate sit for 2 min to allow beads to collect. You should be able to see the solution become clear.

        3. Aspirate the liquid from each well. Switch tips after each well to avoid cross-contamination.

        4. When aspirating, make sure to hold the plate flush against the magnetic rods to avoid aspirating the beads.

          Note: Adjust the direction depending on where the magnetic rod sits relative to the wells.

        5. After aspirating the liquid from each row of wells, add 400 μL of PhIP-Seq Wash Buffer to the empty wells to prevent the beads from drying out.

        6. Remove the plate from the magnetic separation rack and use a multichannel pipettor to resuspend the beads in all the wells by pipetting up and down 10 times.

        7. Repeat steps i-iv for a total of three washes. During the first wash, transfer the beads to the new 96-well deep well plate.

        8. Cover the plate with a new MicroAmp optical adhesive seal and centrifuge the beads at 500 × g for 1 min. Aspirate any remaining liquid.

    10. Resuspend the beads in each well in 40 μL of sterile water and transfer to PCR plate. Seal plate.

    11. Spin PCR plate with resuspended beads in centrifuge for ~10 s, until centrifuge reaches 50 × g, to collect beads off the sides of the wells.

    12. Heat plate to 95°C for 10 min to lyse T7 phage.

    13. Store plate at -80°C for up to a week or proceed directly to library preparation for next-generation sequencing.


  4. Library preparation for next-generation sequencing (Video 5)

    Note: When setting up PCRs, keep PCR plate on aluminum block on ice at all times. Keep all reagents on ice at all times.


    Video 5. Library preparation for next-generation sequencing


    1. Thaw frozen beads on ice, then centrifuge at 1,000 × g for 2 min.

    2. Make PCR1 master mix, mix well, and transfer to a reservoir on ice.

      Component Stock concentration Final concentration 1 rxn (μL) 110 rxns (μL)
      Sterile water 2.68 294.8
      Reaction Buffer 6.0 660
      dNTPs 10 mM 0.3 mM 0.90 99
      Primer IS7 100 μM 0.2 μM 0.06 6.6
      Primer IS8 100 μM 0.2 μM 0.06 6.6
      Q5 2 U/μL 0.02 U/μL 0.30 33
      Template 20
      Total 30 μL

    3. Aliquot 10 μL of PCR1 master mix to each well of a new 96-well PCR plate. Keep plate on aluminum block on ice at all times.

    4. Resuspend beads by pipetting and add 20 μL of beads to corresponding wells. Mix well by pipetting.

      Note: If sequencing input library, mix 5 μL of input library and 15 μL of sterile water and use this as the template for the PCR1 instead of 20 μL of resuspended beads.

    5. Spin PCR1 plate in centrifuge for ~10 s, until centrifuge reaches 50 × g, then immediately remove plate and return to aluminum block on ice.

    6. Run PCR1.

      STEP TEMP TIME
      Initial Denaturation 98°C 30 s
      28 Cycles total 98°C 5 s
      66°C 10 s
      72°C 30 s
      Final Extension 72°C 2 min
      Hold 4–10°C

    7. Make PCR2 master mix, mix well, and transfer to reagent reservoir.

      Notes:

      1. Sample multiplexing is achieved using barcoded PCR2 RV primers (Index primers).

      2. Index primers are diluted to 2.5 μM and kept in a 96-well plate.

        Component Stock concentration Final concentration 1 rxn (μL) 110 rxns (μL)
        Sterile water 4.55 500.5
        Reaction Buffer 2.0 220
        dNTPs 10 mM 0.3 mM 0.3 33
        Primer IS4 100 μM 0.5 μM 0.05 5.5
        Index primer 2.5 μM 0.5 μM 2.0
        Q5 2 U/μL 0.02 U/μL 0.1 11
        Template 1.0
        Total 10 μL

    8. Distribute 7 μLof PCR2 master mix to each well of a new 96-well PCR plate.

    9. Add 2 μL of appropriate index primers (diluted to 2.5 μM) to corresponding wells.

    10. Add 1 μL of appropriate PCR1 product to corresponding wells as template.

    11. Mix PCR reactions by running the paddle rapidly across the bottom of PCR plate a few times, thus agitating the wells. Spin PCR plate in centrifuge for ~10 s until centrifuge reaches 50 × g, then immediately return plate to aluminum block on ice.

    12. Run PCR2.

      STEP TEMP TIME
      Initial Denaturation 98°C 30 s
      Eight cycles total 98°C 5 s
      68°C 10 s
      72°C 30 s
      Final Extension 72°C 2 min
      Hold 4–10°C

      Note: Steps 13–19 are quality control steps to verify that there is an amplicon in all appropriate wells.

    13. Dilute PCR2 product 1:40,000 in sterile water.

      1. Serially dilute 2 μL of PCR2 product in 398 μL of sterile water (1:200 dilution) twice.

    14. Make qPCR master mix, mix well, and transfer to reservoir.

      Component Stock concentration Final concentration 1 rxn (μL) 110 rxns (μL)
      Sterile water 8.75 962.5
      Universal Mix 10 1100
      3’ NEST qPCR primer 100 μM 0.5 μM 0.1 11
      5’ NEST qPCR primer 100 μM 0.5 μM 0.1 11
      NEST qPCR probe 100 μM 0.25 μM 0.05 5.5
      PCR2 template, diluted 1:40,000 1.0
      Total 20 μL

    15. Distribute 19 μL of qPCR master mix to each well of a 96-well qPCR plate.

    16. Add 1 μL of appropriate PCR2 product, diluted 1:40,000, to corresponding wells as template.

    17. Mix qPCR reactions by running paddle rapidly across bottom of PCR plate a few times, thus agitating the wells. Spin in centrifuge for ~10 s, until centrifuge reaches 50 × g.

    18. Run qPCR.

      STEP TEMP TIME
      1 Cycle 50°C 2 min
      95°C 10 min
      40 Cycles 95°C 15 s
      60°C 2 min

    19. If a well fails to amplify by qPCR, run out the corresponding PCR1 and PCR2 products to diagnose the problem. If necessary, redo PCR1 and/or PCR2.

    20. Pool 2 μL of each sample of PCR2 in a reservoir, mix, and transfer to 1.5 mL microfuge tube.

      Notes:

      1. Pool samples from individual plates separately.

      2. If sequencing input library, add 10× the volume of any given sample into the final pool, i.e., if pooling 2 μL of each sample, add 20 μL of the input library to the pool.

    21. Run 40 μL of pooled PCR2 products from each plate on a 2% agarose TAE gel.

    22. Gel extract correct size band using QIAgen Gel Extraction Kit.

      Notes:

      1. The expected amplicon size for the T7 VirScan library is 376 bp.

      2. There may be a faint band directly below the correct-size band. Do not extract this faint lower band, as it contains products with truncated or missing Primer IS4 or Index Primer sequences.

    23. PCR purify gel-extracted samples using QIAquick PCR Purification kit.

      Note: PCR purification is performed after gel extraction to ensure greater purity of the sample prior to next-generation sequencing.

    24. Quantitate DNA using dsDNA HS Qubit assay, then pool equal amounts (ng) of each plate.


  5. Next-generation sequencing

    1. Submit the pooled library for sequencing. The following sequencing parameters are required:

      Note: We generally sequence pooled libraries of 192 samples at a core facility with an Illumina NextSeq 500 instrument and the NextSeq 500/550 High Output Kit v2.5 (75 Cycles), which yields ~400M reads. We order single-read, single-index sequencing, detailed below.

      1. Read 1: 75 cycles

        Note: Only 50 cycles are required, but we typically order 75 cycles and truncate the reads during the Data Analysis steps.

      2. Index I7: 7 cycles

      3. Sequencing depth: 1M reads/sample.

      4. Custom sequencing primer for Read 1: T7-Illumina-READ1-A

Data analysis

Notes:

  1. In the instructions below, lines of code are bolded. These instructions are for use on a computing cluster using SLURM.

  2. Example VirScan data for two serum samples and their technical replicates are provided (Supplementary materials). Data files include a sample legend, fastq files, BAM files, alignment report files, indexed BAM files, counts files, count.combined files (counts summed across four lanes of a Nextseq 500 flow cell), a counts table (count.combined data presented in a table format; summed counts for no-serum controls are provided in the column “input”), a Z-score table (again, summed counts for no-serum controls are present in the column ‘input’), a hits_combined table, and virus scores files.


  1. Align sequencing reads to a reference file

    1. Use the reference fasta file for the VirScan library (“vir3.fasta”) (Supplementary materials) and generate index files with the .ebwt extension. Run the following commands:

      module load gcc/6.2.0


      module load bowtie/1.2.2


      bowtie-build vir3.fasta vir3

    2. Align sequencing reads to the reference file. See “script.align.sh” and edit as needed (Supplementary materials). The output file is a file that ends in “.bam”

      Notes:

      1. Sequencing reads are typically distributed as fastq files. These fastq files are stored in a subdirectory called “raw.data”.

      2. In “script.align.sh”, “bowtie -3 25” trims 25 nucleotides off the 3’ end of each sequencing read. This is done if sequencing reads are 75 nucleotides in length. The reference file only includes the first 50 nucleotides of each member of the library, so the sequencing reads must be trimmed down to 50 nucleotides to align correctly to the reference.

      3. In “script.align.sh”, replace “path_to_vir3_reference_fasta_and_index_files” with the appropriate path.

      ./ script.align.sh

    3. Check the alignment report file that ends in “.out”

      Note: Typically, >85% of the reads align to the reference file.

    4. Index files with the following commands. The output file is a file that ends in “.bai”

      module load gcc/6.2.0


      module load samtools/1.3.1


      for i in raw.data/*.bam; do samtools index $i; done

    5. Count indexes with the following commands. The output is a file that ends in “.count.csv”

      module load gcc/6.2.0


      module load samtools/1.3.1


      for i in raw.data/*.bam; do samtools idxstats $i | cut -f 1,3 | sed -e '/^\*\t/d' -e '1 i id\tSAMPLE_ID' | tr "\\t" "," >${i%.bam}.count.csv; done

    6. Gzip the counts files with the following command.

      for i in raw.data/*.csv; do gzip $i; done

    7. Create a directory called “log_directory” with the following command.

      mkdir log_directory
    1. If the same sample is run on two or more lanes of a flow cell and separate files are provided for each flow cell, combine the counts files from the different lanes using the following commands. These commands require the python script “combine_two_lanes.py” to be copied to the folder where you are running the commands (Supplementary materials).

      Note: In the code below, the samples were run on four lanes of an Illumina Nextseq 500 flow cell. The suffix of each count file is “L001_R1_001.count.csv.gz” if the count file was from the first lane of the flow cell, “L002_R1_001.count.csv.gz” if the count was from the second lane of the flow cell, etc.

      module load gcc/6.2.0


      module load python


      for i in raw.data/*L001_R1_001.count.csv.gz; do python combine_two_lanes.py $i ${i%1_R1_001.count.csv.gz}2_R1_001.count.csv.gz ${i%1_R1_001.count.csv.gz}1_2_R1_001.count.csv; done


      for i in raw.data/*L003_R1_001.count.csv.gz; do python combine_two_lanes.py $i ${i%3_R1_001.count.csv.gz}4_R1_001.count.csv.gz ${i%3_R1_001.count.csv.gz}3_4_R1_001.count.csv; done


      for i in raw.data/*L001_2_R1_001.count.csv; do python combine_two_lanes.py $i ${i%1_2_R1_001.count.csv}3_4_R1_001.count.csv ${i%1_2_R1_001.count.csv}1_2_3_4_R1_001.count.combined.csv; done


    2. Gzip the count.combined files with the following command.

      for i in raw.data/*1_2_3_4_R1.count.combined.csv; do gzip $i; done


  2. Calculate Z-scores

    Note: To perform the Z-score analysis, count.combined files are merged into a table, and columns corresponding with no-serum controls are summed in a column called “input”.

    1. Edit the R script “Zscore_analysis.R” to include the path to the count.combined table file and the desired path to the output file, then run the script (Supplementary materials). The packages “mmR_0.1.0” and “virScanR_0.1.0.9000” are required (Supplementary materials).

      Note: The file “Zscores_vir3” contains the results after this step (Supplementary materials).

    2. A Z-score of at least 3.5 in both technical replicates of a sample is required to call a peptide a “hit”.

      Note: The file “hits_combined_vir3_3.5_cutoff” contains the results after this step (Supplementary materials).


  3. Calculate virus scores

    1. Create a directory called “hits”. In this directory should be .csv files for each sample with “True” or “False” values for each peptide ID, depending on whether the peptide scored as a hit (Z-score > 3.5) in both technical replicates of a sample or not. These files may be created by splitting each column of the “hits_combined_vir3_3.5_cutoff” file into a separate files (Supplementary materials).

    2. Generate virus scores files using the following code:

      Note: The “VIR3_clean” file provides the annotations for the oligos” (Supplementary materials). There are 115,753 oligos in the Vir3 library. Some protein fragments are identical in different viruses, and in these case there are multiple rows in the “VIR3_clean” file that correspond to a single oligo. To identify the viral source of a given peptide, look for the row(s) in the VIR3_clean file with the "id" value of the given peptide.


      for i in hits/*.csv.gz; do python calc_scores_nofilter.py $i VIR3_clean.csv.gz Species 7 >virus_scores_$i; done


  4. Determining virus seropositivity

    1. A sample is determined to be seropositive for a virus if the virus_score > VirScan_viral_threshold and if at least one public epitope from that virus scores as a hit. The file “VirScan_viral_thresholds” contains the thresholds for each virus (Supplementary materials).

      Note: Public epitope annotations are available upon request.

Recipes

  1. Phage extraction buffer

    20 mM Tris-HCl, pH 8.0

    100 mM NaCl

    6 mM MgSO4

    Store at 4°C

  2. PhIP-Seq Wash Buffer

    50 mM Tris-HCl, pH 7.5

    150 mM NaCl

    0.1% NP-40

    Store at 4°C

Acknowledgments

Funding: E.L.S. was supported by the NSF Graduate Research Fellowship Program. S.J.E. is an Investigator with the Howard Hughes Medical Institute.

Original research papers from which this protocol was derived: Larman et al. (2011), Xu et al. (2015), and Mina et al. (2019).

We thank A. Kohlgruber for designing the schematic in the graphical abstract.

Competing interests

S.J.E. is a founder of TSCAN Therapeutics, MAZE Therapeutics, Mirimus, and ImmuneID. S.J.E. serves on the scientific advisory board of Homology Medicines, TSCAN Therapeutics, MAZE Therapeutics, XChem, and is an advisor for MPM, none of which impact this work. E.L.S. was a consultant for ImmuneID. S.J.E. is an inventor on a patent application filed by the Brigham and Women's Hospital (US20160320406A) that covers the use of the VirScan library to identify pathogen antibodies in blood.

Ethics

Human specimens were collected in accordance with the local protocol governing human research after obtaining informed written consent from the donors. Secondary use of all human samples for the purposes of this work was exempted by the Brigham and Women’s Hospital Institutional Review Board (protocol number 2013P001337).

References

  1. Bjornevik, K., Cortese, M., Healy, B. C., Kuhle, J., Mina, M. J., Leng, Y., Elledge, S. J., Niebuhr, D. W., Scher, A. I., Munger, K. L., et al. (2022). Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375(6578): 296-301.
  2. Borriello, F., Poli, V., Shrock, E., Spreafico, R., Liu, X., Pishesha, N., Carpenet, C., Chou, J., Di Gioia, M., McGrath, M. E., et al. (2022). An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity. Cell 185(4): 614-629 e621.
  3. Chen, G., Shrock, E. L., Li, M. Z., Spergel, J. M., Nadeau, K. C., Pongracic, J. A., Umetsu, D. T., Rachid, R., MacGinnitie, A. J., Phipatanakul, W., et al. (2021). High-resolution epitope mapping by AllerScan reveals relationships between IgE and IgG repertoires during peanut oral immunotherapy. Cell Rep Med 2(10): 100410.
  4. Garrett, M. E., Itell, H. L., Crawford, K. H. D., Basom, R., Bloom, J. D. and Overbaugh, J. (2020). Phage-DMS: A Comprehensive Method for Fine Mapping of Antibody Epitopes. iScience 23(10): 101622.
  5. Hill, J. A., Krantz, E. M., Hay, K. A., Dasgupta, S., Stevens-Ayers, T., Bender Ignacio, R. A., Bar, M., Maalouf, J., Cherian, S., Chen, X. et al. (2019). Durable preservation of antiviral antibodies after CD19-directed chimeric antigen receptor T-cell immunotherapy. Blood Adv 3(22): 3590-3601.
  6. Kosuri, S., Eroshenko, N., Leproust, E. M., Super, M., Way, J., Li, J. B. and Church, G. M. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28(12): 1295-1299.
  7. Langmead, B., Trapnell, C., Pop, M. and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3): R25.
  8. Larman, H. B., Zhao, Z., Laserson, U., Li, M. Z., Ciccia, A., Gakidis, M. A., Church, G. M., Kesari, S., Leproust, E. M., Solimini, N. L. et al. (2011). Autoantigen discovery with a synthetic human peptidome. Nat Biotechnol 29(6): 535-541.
  9. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R. and Genome Project Data Processing, S. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16): 2078-2079.
  10. Mandel-Brehm, C., Dubey, D., Kryzer, T. J., O'Donovan, B. D., Tran, B., Vazquez, S. E., Sample, H. A., Zorn, K. C., Khan, L. M., Bledsoe, I. O., et al. (2019). Kelch-like Protein 11 Antibodies in Seminoma-Associated Paraneoplastic Encephalitis. N Engl J Med 381(1): 47-54.
  11. Mina, M. J., Kula, T., Leng, Y., Li, M., de Vries, R. D., Knip, M., Siljander, H., Rewers, M., Choy, D. F., Wilson, M. S., et al. (2019). Measles virus infection diminishes preexisting antibodies that offer protection from other pathogens. Science 366(6465): 599-606.
  12. Mohan, D., Wansley, D. L., Sie, B. M., Noon, M. S., Baer, A. N., Laserson, U. and Larman, H. B. (2018). PhIP-Seq characterization of serum antibodies using oligonucleotide-encoded peptidomes. Nat Protoc 13(9): 1958-1978.
  13. Pou, C., Nkulikiyimfura, D., Henckel, E., Olin, A., Lakshmikanth, T., Mikes, J., Wang, J., Chen, Y., Bernhardsson, A. K., Gustafsson, A., et al. (2019). The repertoire of maternal anti-viral antibodies in human newborns. Nat Med 25(4): 591-596.
  14. R Core Team. (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ”https://www.r-project.org/”.
  15. Schubert, R. D., Hawes, I. A., Ramachandran, P. S., Ramesh, A., Crawford, E. D., Pak, J. E., Wu, W., Cheung, C. K., O'Donovan, B. D., Tato, C. M., et al. (2019). Pan-viral serology implicates enteroviruses in acute flaccid myelitis. Nat Med 25(11): 1748-1752.
  16. Shrock, E., Fujimura, E., Kula, T., Timms, R. T., Lee, I. H., Leng, Y., Robinson, M. L., Sie, B. M., Li, M. Z., Chen, Y., et al. (2020). Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Science 370(6520).
  17. Smith, G. P. and Petrenko, V. A. (1997). Phage Display. Chem Rev 97(2): 391-410.
  18. Xu, G. J., Kula, T., Xu, Q., Li, M. Z., Vernon, S. D., Ndung'u, T., Ruxrungtham, K., Sanchez, J., Brander, C., Chung, R. T., et al. (2015). Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome. Science 348(6239): aaa0698.
  19. Zamecnik, C. R., Rajan, J. V., Yamauchi, K. A., Mann, S. A., Loudermilk, R. P., Sowa, G. M., Zorn, K. C., Alvarenga, B. D., Gaebler, C., Caskey, M., et al. (2020). ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Rep Med 1(7): 100123.

简介

[摘要]分析抗体的特异性可以揭示有关体液免疫反应及其靶向抗原的大量信息。在这里,我们提出了VirScan的协议,这是一种噬菌体免疫沉淀测序 ( PhIP -Seq) 方法的应用,用于分析人类抗病毒抗体的特异性。 伴随该协议的是实验过程的视频。 VirScan以及更普遍的PhIP -Seq 是通过将高通量 DNA 寡核苷酸合成和噬菌体展示与下一代测序相结合来实现高通量抗体分析的技术。在VirScan方法中,人类血清样本针对跨越整个人类病毒蛋白质组的肽库进行筛选。结合的噬菌体被免疫沉淀和测序,识别抗体识别的病毒肽。 VirScan是一个强大的工具,用于揭示个体病毒暴露历史、绘制感兴趣病毒的表位图谱以及研究病毒免疫的基本机制。

图形概要:


[背景] VirScan ( Xu et al. , 2015 ) 基于称为噬菌体免疫沉淀测序 ( PhIP -Seq) ( Larman ) 的通用技术 等。 , 2011;莫汉等人。 , 2018;曼德尔-布雷姆等人。 ,2019 年,加勒特等人,2020 年)。在PhIP -Seq 中,设计了一个蛋白质组规模的肽库,编码这些肽的 DNA 寡核苷酸被合成并克隆到 T7 噬菌体展示系统中。每个噬菌体在其基因组中编码一种肽的序列,并在其表面展示相同的肽,从而将基因型与表型联系起来(Smith 和Petrenko ,1997; Kosuri 等。 , 2010)。对于每个PhIP -Seq 反应,噬菌体展示文库与含有人抗体的样品混合,抗体与噬菌体表面上的相关表位结合。然后噬菌体-抗体复合物被免疫沉淀,未结合的噬菌体被洗掉。来自结合噬菌体的插入 DNA 的 PCR 扩增和高通量测序揭示了样品中抗体靶向的肽。 最初的PhIP -Seq 分析是使用源自人类蛋白质组的肽的噬菌体展示库来检测自身抗体,而 VirScan使用源自人类病毒组的肽库来识别针对病毒抗原的抗体的特异性。
Mohan等人提供了PhIP -Seq 相对于肽或蛋白质微阵列用于高通量表位分析的优缺点比较。 (2018 年) 。简而言之,相对于肽和全蛋白微阵列, PhIP -Seq 能够实现更高的通量、更便宜和更高度可编程的分析。与蛋白质微阵列相比, PhIP -Seq的一个缺点是实验过程需要更长的时间来执行,因为它的读数涉及下一代测序。与所有基于肽的表位分析方法一样, PhIP - Seq 通常不能检测不连续的表位或涉及翻译后修饰的表位。
一篇描述 PhIP-Seq 协议的综合文章已经发表( Mohan等人,2018 年),对于那些对使用VirScan技术感兴趣的人来说,这是一个宝贵的资源。这篇文章通过展示实验方案的视频(视频1-5 )和包括与 VirScan 特定数据分析相关的信息(补充材料)来补充该资源。该协议不包含与VirScan噬菌体展示库的设计和生成相关的信息,因为Mohan等人已深入介绍了这些方法。 (2018)和徐等人。 (2015 年) 。本文假设研究人员可以访问VirScan文库,并重点关注下游实验程序,即噬菌体-抗体复合物形成、免疫沉淀和制备用于下一代测序的 DNA 文库,视频中也有描述(视频1-5 ) 。此外,该协议涵盖VirScan特定的数据分析步骤,包括病毒计算命中、病毒评分计算和病毒血清阳性测定。
VirScan协议可以通过补充文库、替代免疫沉淀试剂和人血清以外的输入样本进行修改,以解决更广泛的科学问题。可以设计丙氨酸扫描和饱和诱变文库以实现抗体表位的高分辨率作图,如Shrock等人所进行的。 (2020)和陈等人。 (2021 年) 。标准免疫沉淀试剂,蛋白 A 和蛋白 G,可以用同种型特异性二抗代替,以分析除 IgG 之外的抗体同种型,如 IgA 或IgE ,如Shrock等人中所述。 (2020)和陈等人。 (2021 年) 。该方案可用于除人类以外的几种哺乳动物物种的血清样本,包括小鼠和非人类灵长类动物,因为蛋白 A 和蛋白 G 与小鼠和非人类灵长类动物 IgG 以及人类 IgG 结合( Borriello 等。 , 2022)。最后,除血清之外的含抗体样品,包括唾液、母乳和培养的 B 细胞上清液,可用作VirScan的输入样品。
VirScan已成功用于许多应用,包括估计个体接触过的病毒种类的数量( Xu et al. , 2015);表明麻疹病毒感染会减少先前存在的抗体库,使个体容易再次感染病原体( Mina等人,2019 年);研究人类婴儿的母源抗体库 ( Pou 等。 , 2019);研究针对 CD19 的 CART 疗法对抗病毒抗体库的影响(Hill等,2019);揭示罕见神经系统疾病急性弛缓性脊髓炎的推定病毒病因( Schubert等人,2019 年),以高分辨率绘制 SARS-CoV-2 线性表位并确定 COVID-19 严重程度的体液免疫相关性( Shrock等人。 , 2020;扎梅尼克 等。 , 2020 年),并提供证据证明 Epstein-Barr 病毒感染会增加随后发展为多发性硬化症的风险 ( Bjornevik 等。 , 2022)。

关键字:VirScan, PhIP-Seq, 噬菌体展示技术, 合成生物学, 高通量筛选, 血清学, 抗体, 表位, 病毒, 免疫学



材料和试剂


1. 移液器吸头 SR LTS 20 µL F 960A/5( Rainin ,目录号:17005860),储存温度:室温
2. 移液器吸头 SR LTS 200 µL F 960A/5( Rainin ,目录号:17005859),储存温度:室温
3. 移液器吸头 SR LTS 1,200 µL F 768A/4( Rainin ,目录号:17007084),储存温度:室温
4. 一次性血清移液管,聚苯乙烯,无菌,堵塞,5 mL (例如, VWR ,目录号: 89130-896 ),储存温度:室温
5. 一次性血清移液管,聚苯乙烯,无菌,堵塞,10 mL (例如, VWR ,目录号: 89130-898 ),储存温度:室温
6. 一次性血清移液管,聚苯乙烯,无菌,堵塞,25 mL (例如, VWR ,目录号: 89130-900 ),储存温度:室温
7. 一次性血清移液管,聚苯乙烯,无菌,堵塞,50 mL (例如, VWR ,目录号: 89130-902 ),储存温度:室温
8. 试剂容器,无菌(例如, Corning,Costar,目录号:4870),储存温度:室温
9. 无菌过滤器储存瓶/接收器(例如, Thermo Fisher, Nalgene,目录号:455-0500),储存温度:室温
10. 深孔板,96 孔,PP,1.1 mL,标准,U 形底(Cole-Parmer, BrandTech ,目录号:EW-07904-04),储存温度:室温
11. Kimtech Science Kimwipes Delicate Task Wipes(Kimberly-Clark,目录号:34155),储存温度:室温
12. 密封桨( USA Scientific,目录号: 2928-7355),储存温度:室温 
13. MicroAmp光学胶膜(Thermo Fisher,Applied Biosystems,目录号: 4311971),储存温度:室温
14. 彩色标签胶带,Rainbow Pack ( Fisher Scientific, Fisherbrand ,目录号: 15-901-10R),储存温度:室温
15. PCR板,96孔(例如, VWR,目录号: 82006-704),储存温度:室温
16. Bravo Lab 一次性移液器吸头(Agilent,目录号:19477-022),储存温度:室温 
注意:如果使用 Agilent Bravo 执行磁珠清洗,这些是必需的。
17. Nunc 96 孔聚丙烯DeepWell储存板,无菌(Thermo Fisher,Thermo Scientific,目录号:260251),储存温度:室温
18. Nalgene 一次性聚丙烯机器人储存器,无菌(Thermo Fisher,Thermo Scientific,目录号: 1200-1301),储存温度:室温 
注意:如果使用 Agilent Bravo 执行磁珠清洗,这些是必需的。
19. 康宁 96孔透明V型底2 mL聚丙烯深孔板,无菌(Corning,目录号:3960),储存温度:室温
(注意:如果使用 Agilent Bravo 执行磁珠清洗,这些是必需的。
20. MicroAmp快速光学 96 孔反应板,0.1 mL (Thermo Fisher,Applied Biosystems,目录号: 4346906 ),储存温度:室温
(注意:如果使用 Applied Biosystems Fast 7500 系统执行 qPCR,这些是必需的。
21. Qubit Assay Tubes ( Thermo Fisher,Invitrogen,目录号: Q32856),储存温度:室温
22. (可选)IgG(总)人ELISA试剂盒(例如, Thermo Fisher,Invitrogen,目录号: BMS2091),储存温度: 4 °C
23. Tris 缓冲盐水与 Tween 20(TBST-10X)(Cell Signaling,目录号:9997),储存温度:室温
24. 牛血清白蛋白(BSA)( VWR ,目录号:0332-500G),储存温度:4 °C
25. PBS,pH 7.4(例如, Thermo Fisher,目录号:10010023 ),储存温度:室温
26. VirScan T7 噬菌体展示文库(可根据要求提供,储存温度:-80 °C)
注意:基于 T7Select 包装套件(Millipore-Sigma,目录号:70014)存储温度: -80 °C。
27. UltraPure 1M Tris-HCI,pH 8.0 ( Thermo Fisher,Invitrogen,目录号: 15568025 ) ,储存温度:4 °C
28. NaCl(5 M),无RNase ( Thermo Fisher,Invitrogen,目录号: AM9759),储存温度:室温
29. 硫酸镁溶液(Millipore Sigma,目录号: M3409-100ML),储存温度:室温
30. 氯霉素(Millipore Sigma,目录号: C0378-100G ) ,储存温度:粉末室温或-20 ℃重构溶液
31. 霉素B硫酸盐(Millipore Sigma,目录号: B5264-250MG ) ,储存温度:粉末和重构溶液的-20 °C
32. NP-40 Surfact -Amps 洗涤剂溶液( Thermo Fisher,目录号: 85124),储存温度:室温
33. Dynabeads蛋白A(Thermo Fisher,Invitrogen,目录号: 10008D),储存温度:4 °C
34. Dynabeads Protein G(Thermo Fisher,Invitrogen,目录号: 10009D),储存温度:4 °C
35. UltraPure 1 M Tris-HCI 缓冲液,pH 7.5 ( Thermo Fisher,Invitrogen,目录号:15567027) ,储存温度:4 °C
36. UltraPure DNase/RNase-Free Distilled Water ( Thermo Fisher,Invitrogen,目录号: 10977023),储存温度:室温
37. Q5热启动高保真DNA聚合酶( New England Biolabs,目录号: M0493L)储存温度:-20 °C
38. dNTP Set(100 mM) ( Thermo Fisher,Invitrogen,目录号: 10297018),储存温度:-20 °C
39. TaqMan Gene Expression Master Mix(Thermo Fisher,Applied Biosystems ,目录号:4369016 ),储存温度: 4 °C
40. UltraPure Agarose (Thermo Fisher, Invitrogen,目录号: 16500100),储存温度:室温
41. UltraPure DNA Typing Grade 50 × TAE Buffer (Thermo Fisher,Invitrogen,目录号: 24710030),储存温度:室温
42. QIAquick Gel Extraction Kit(250)(QIAGEN,目录号:28706 ),储存温度:室温
43. QIAquick PCR Purification Kit(250)(QIAGEN,目录号:28106 ),储存温度:室温
44. Qubit dsDNA HS Assay Kit ( Thermo Fisher,Invitrogen,目录号: Q32851),储存温度:混合,室温和4 °C
45. HPLC纯化的引物(IDT ,储存温度:-20 °C )


引物名称 引物序列 (5' – 3')
IS7 ACACTCTTTCCCTACACGACTCCAGTCAGGTGTGATGCTC
IS8 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCGAGCTTATCGTCGTCATCC
IS4 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACTCC AGT
索引引物 CAAGCAGAAGACGGCATACGAGATNNNNNNNGTGACTGGAGTTTCAGACGTGT
5'NEST-qPCR TCGGGGATCCAGGAATTC
3'NEST-qPCR CGTCGTCATCCTTGTAATCG
NEST_probe /56-FAM/TAATCGCGG/ZEN/CCGCAAGCTTGTC/3IABkFQ/
T7-Illumina-READ1-A TGCTCGGGGATCCAGGAATTCCGCTGCGT
注意:索引引物的正交 7 nt条形码可根据要求提供。


46. 噬菌体提取缓冲液(配方如下) ,储存温度:4 °C
47. PhIP -Seq 洗涤缓冲液(配方如下) ,储存温度:4 °C


设备


1. Pipet-Lite Multi Pipette L12-20XLS+( Rainin ,目录号:17013808)
2. Pipet-Lite Multi Pipette L12-200XLS+( Rainin ,目录号:17013810)
3. E4 Pipette Multi E12-1200XLS+( Rainin ,目录号:17014499)
4. 便携式 Pipet-Aid XP 移液器控制器(Drummond,目录号:4-000-101)
5. 旋转器(例如,Barnstead/ Thermolyne ,型号:415110 )
6. 带有摆桶转子组件和微孔板载体的台式离心机(例如Beckman Coulter,型号: Allegra X-15R 或 Avanti J-15R,摆桶转子组件:SX4750A 或 JS-4. 750, 微孔板载体: SX4750 )
7. Bravo NGS 自动化液体处理平台(Agilent,目录号: G5573AA)
8. 96孔微量滴定板磁分离架(NEB,目录号: S1511S )
注意:如果手动执行磁珠清洗,这是必要的。
9. 热循环仪(例如, Bio-Rad,型号:C1000 Touch with 96-well Fast Reaction Module,目录号:1851196)
10. 用于 0.2 mL 管的 96 孔铝块( Universal Medical,目录号:81001 )
11. 7500 Fast Dx Real-Time PCR Instrument,带笔记本电脑(Thermo Fisher,Applied Biosystems,目录号:4406984)
12. Qubit 4荧光计( Thermo Fisher,Invitrogen,目录号:Q33238 )


软件 


1. 领结(Langmead等人,2009 年)
2. samtools (Li et al. , 2009)
3. python( Python 软件基金会。Python 语言参考,2.7 版。可在http://www.python.org获得)
4. gcc (GNU 编译器集合,版本 6.2.0。文档位于https://gcc.gnu.org/onlinedocs/gcc.pdf )
5. R (R 核心团队,2017 年)


程序


 
视频 1. 简介


A. 挡板(视频2)


 
视频 2. 挡板


1. 为每个 96 孔深孔板 ( Cole-Parmer )准备 100 m L 的 TBST 3% BSA。转移到试剂容器。
2. 在 96 孔深孔板的每个孔中加入 1 mL 的 TBST 3% BSA。
3. 用 kimwipes 吸干板顶部以去除多余的液体。
4. MicroAmp光学胶膜密封板。
注意:可以通过从板的中心向外按压以消除大气泡来密封板,然后使用密封桨消除所有剩余的气穴。
5. 反转板数次以确保液体在整个板中移动。
6. °C 下将 96 孔深孔板粘贴到板旋转器上,并在一夜之间端到端旋转。


B. 噬菌体抗体复合物的形成(视频 3 )


 
视频 3. 噬菌体抗体复合物的形成


1. 在冰上解冻VirScan T7 噬菌体文库。文库的特征如表 1 所示。
2. 为每个 96 孔深孔板准备 110 mL 的稀释 T7 噬菌体库。有关如何准备稀释的噬菌体库的信息,请参见表 2。
3. 混合得很好。


表1 。 VirScan T7 噬菌体文库特征
复杂性(Vir3 版本) 115,753 名成员
所需的最终浓度 2 × 10 5 pfu/mL 每个文库成员,或大约 2 × 10 10 pfu/mL


表 2. 制备稀释的噬菌体文库
零件 库存集中度 最终浓度 110次
VirScan噬菌体文库 9.9 × 10 10 pfu/mL(可能因批次而异) 2 × 10 10 pfu/mL 22.2 毫升
氯霉素 50 毫克/毫升 (1,000 × ) 50微克/毫升(1 × ) 110微升
卡那霉素 50 毫克/毫升 (1,000 × ) 50微克/毫升(1 × ) 110微升
噬菌体提取缓冲液 至 110 毫升


4. 在 96 孔 PCR 板中的 PBS 中将血清等分试样稀释至 0.2 μg / μL 。
笔记:
a. 中IgG的浓度一般为5-10微克/微升。在 98 μL PBS(1:50 稀释)中稀释 2 μL血清,以达到约 0.2 μg / μL人 IgG的浓度。搅拌均匀。
b. 如果需要,可以通过 IgG ELISA 测量样品中 IgG 的浓度。
c. 血清样本通常一式两份运行。
d. 每次运行通常包括八个无血清对照。
5. 将 96 孔深孔板中的封闭溶液倒入水槽中。轻弹板数次以去除所有阻塞溶液。
6. 用kimwipe 涂抹板表面以去除液体。
7. 在每口井中加入 1 mL 的稀释噬菌体库。
8. 用kimwipe 涂抹板表面以去除多余的液体。
9. μg IgG 的血清加入每孔中,或从先前制备的 0.2 μg / μL板中加入10 μL 。
10. 用kimwipe 涂抹板表面以去除多余的液体。
11. 使用桨,用新的MicroAmp光学胶膜密封板非常好。确保孔之间没有气泡。
12. 反转板数次以确保液体在整个板中移动。在 4°C 下将板固定在旋转器上,并通过端到端混合旋转 20 小时或过夜。
13. 用稀释的血清样品密封板并储存在-80 °C。


C. 免疫沉淀(视频 4)


 
视频 4. 免疫沉淀


1. 将 96 孔深孔板以 500 × g离心3 分钟,以收集远离密封的液体。
2. 卸下密封件时,紧紧按住板。避免孔之间的飞溅和交叉污染。
3. 重悬 Protein A 和 Protein G Dynabeads ,直到底部没有剩余的珠子沉淀。
4. 对于每个 96 孔深孔板,将 2 mL 蛋白 A 和 2 mL 蛋白 G Dynabeads添加到试剂库中,并与血清移液器混合。
5. 添加 40 μL 蛋白 A/G 到 96 孔深孔板的每个孔中。
6. 用kimwipe 涂抹板表面以去除多余的液体。
7. 密封板用新的MicroAmp光学粘合剂密封,并用胶带将板固定在旋转器上。在室温下旋转 4 小时或在 4°C 下过夜。
8. 一次取下一个盘子进行清洗。将 96 孔深孔板以 500 × g离心3 分钟,以收集远离密封的液体。
9. 使用液体处理机器人执行三次洗涤,如视频 4 所示,每次用 170 μL的PhIP -Seq 洗涤缓冲液洗涤。在洗涤过程中的某一时刻,将珠子转移到新的 96 孔深孔板(Thermo Fisher)中,以避免可能与原始板的孔非特异性结合的噬菌体。 Bravo 协议文件可在补充材料中找到。
a. 或者,手动执行清洗。
i. 将板放在磁分离架上。
ii. 让板静置 2 分钟以收集珠子。您应该能够看到解决方案变得清晰。
iii. 从每个孔中吸出液体。在每口井后切换提示以避免交叉污染。
iv. 抽吸时,请确保将板与磁棒齐平,以避免吸出珠子。
注意:根据磁棒相对于孔的位置调整方向。
v. 从每排孔中吸出液体后,在空孔中加入 400 μL PhIP - Seq 洗涤缓冲液,以防止珠子变干。
vi. 从磁分离架上取下盘子,使用多通道移液器通过上下移液 10 次重新悬浮所有孔中的珠子。
vii. 重复步骤i -iv 总共洗涤三次。在第一次洗涤期间,将珠子转移到新的 96 孔深孔板中。
viii. 用新的MicroAmp光学胶封盖住盘子,并以 500 × g的速度将珠子离心1 分钟。吸出任何剩余的液体。
10. 将每口井中的珠子重新悬浮在 40 μL的无菌水中并转移到 PCR 板中。密封板。
11. 用重悬珠子在离心机中旋转 PCR 板约 10 秒,直到离心机达到 50 × g ,从孔的侧面收集珠子。
12. 将板加热至 95 °C 10 分钟以裂解 T7 噬菌体。
13. 板在 -80 °C 下储存长达一周或直接进行库准备以进行下一代测序。


D. 为下一代测序准备文库(视频 5)
注意:设置 PCR 时,始终将 PCR 板放在冰上的铝块上。始终将所有试剂放在冰上。


 
视频 5。 用于下一代测序的文库制备


1. 在冰上解冻冷冻珠子,然后以 1,000 × g离心2 分钟。
2. 制作 PCR1 预混液,充分混合,然后转移到冰上的储液罐中。


零件 库存集中度 最终浓度 1次反应( μL ) 110次反应( μL )
无菌水 2.68 294.8
反应缓冲液 5 × 1 × 6.0 660
dNTPs 10毫米 0.3 毫米 0.90 99
底漆 IS7 100 微米_ 0.2微米_ 0.06 6.6
底漆 IS8 100微米_ 0.2微米_ 0.06 6.6
Q5 2 U/ μL 0.02 U/ μL 0.30 33
模板 2 × 1 × 20
全部的 30微升


3. 将 10 μL的 PCR1 主混合物等分到新的 96 孔 PCR 板的每个孔中。始终将盘子放在冰上的铝块上。
4. 通过移液重悬珠子,并将 20 μL的珠子添加到相应的井中。通过移液充分混合。
注意:如果对输入库进行排序,请混合 5 μL的输入库和 15 μL的无菌水,并将其用作 PCR1 的模板,而不是 20 μL的重悬珠。
5. 在离心机中旋转 PCR1 板约 10 秒,直到离心机达到 50 × g ,然后立即取出板并放回冰上的铝块。
6. 运行 PCR1。
步 温度 时间
初始变性 98°C 30 秒
总共 28 个周期 98°C 5 秒
66°C 10 秒
72°C 30 秒
最终扩展 72°C 2 分钟
抓住 4–10°C


7. 制作 PCR2 预混液,充分混合,然后转移到试剂槽中。
笔记:
a. 使用带条形码的 PCR2 RV 引物(索引引物)实现样品多路复用。
b. 将索引引物稀释至 2.5 μM并保存在 96 孔板中。


零件 库存集中度 最终浓度 1次反应( μL ) 110次反应( μL )
无菌水 4.55 500.5
反应缓冲液 5 × 1 × 2.0 220
dNTPs 10毫米 0.3 毫米 0.3 33
底漆 IS4 100微米_ 0.5微米_ 0.05 5.5
索引引物 2.5微米_ 0.5微米_ 2.0
Q5 2 U/ μL 0.02 U/ μL 0.1 11
模板 2 × 1 × 1.0
全部的 10微升


8. 7 μL的PCR2 主混合物分配到新的 96 孔 PCR 板的每个孔中。
9. 添加 2 μL 将适当的索引引物(稀释至 2.5 μ M )加入相应的孔中。
10. 将 1 μL适当的 PCR1 产品添加到相应的井中作为模板。
11. 通过在 PCR 板底部快速运行桨几次来混合 PCR 反应,从而搅动孔。在离心机中旋转 PCR 板约 10 秒,直到离心机达到 50 × g ,然后立即将板放回冰上的铝块中。
12. 运行 PCR2。


步 温度 时间
初始变性 98°C 30 秒
八个周期 98°C 5 秒
68°C 10 秒
72°C 30 秒
最终扩展 72°C 2 分钟
抓住 4–10°C


注意:步骤 13 – 19 是质量控制步骤,用于验证所有合适的孔中是否存在扩增子。
13. 在无菌水中稀释 PCR2 产物 1:40,000。
a. 连续稀释 2 μL PCR2 产物 在 398 μL无菌水(1:200 稀释)中两次。
14. 制作 qPCR 主混合物,混合均匀,然后转移到水库。


零件 库存集中度 最终浓度 1次反应( μL ) 110次反应( μL )
无菌水 8.75 962.5
通用混合 2 × 1 × 10 1100
3' NEST qPCR 引物 100微米_ 0.5微米_ 0.1 11
5' NEST qPCR 引物 100微米_ 0.5微米_ 0.1 11
NEST qPCR 探针 100微米_ 0.25微米_ 0.05 5.5
PCR2 模板,1:40,000 稀释 1.0
全部的 20微升


15. 19 μL的qPCR主混合物分配到 96 孔 qPCR 板的每个孔中。
16. 将 1 μL适当的 PCR2 产物稀释 1:40,000 添加到相应的孔中作为模板。
17. 通过在 PCR 板底部快速运行桨几次来混合 qPCR 反应,从而搅动孔。在离心机中旋转约 10 秒,直到离心机达到 50 × g 。
18. 运行 qPCR。
步 温度 时间
1 个周期 50°C 2 分钟
95°C 10 分钟
40 次循环 95°C 15 秒
60°C 2 分钟


19. 如果孔无法通过 qPCR 扩增,则用完相应的 PCR1 和 PCR2 产物来诊断问题。如有必要,重做 PCR1 和/或 PCR2。
20. 2 μL汇集在水库中,混合并转移到 1.5 mL 微离心管中。
笔记:
a. 分别从单个板中汇集样品。
b. 如果对输入库进行测序,则将任何给定样本的 10倍体积添加到最终池中,即,如果将每个样本合并 2 μL ,则将 20 μL的输入库添加到池中。
21. 在 2% 琼脂糖 TAE 凝胶上从每个板上运行 40 μL的汇集 PCR2 产品。
22. QIAgen Gel Extraction Kit凝胶提取正确大小的条带。
笔记:
a. T7 VirScan文库的预期扩增子大小为 376 bp。
b. 正确大小的带子正下方可能有一条微弱的带子。不要提取这个微弱的较低波段,因为它包含截断或缺失 Primer IS4 或 Index Primer 序列的产品。
23. QIAquick PCR Purification 试剂盒对凝胶提取的样品进行PCR 纯化。
注意:在凝胶提取后进行 PCR 纯化,以确保在下一代测序之前获得更高的样品纯度。
24. 使用 dsDNA HS Qubit 测定法定量 DNA,然后将每个板的等量 (ng) 合并。


E. 新一代测序
1. 提交池库进行排序。需要以下测序参数:
NextSeq 500 仪器和NextSeq 500/550 High Output Kit v2.5( 75 个循环)的核心设施中对 192 个样本的混合文库进行测序, 产生约 4 亿次读取。我们订购单读、单索引测序,详述如下。
a. 读取 1:75 个循环
注意:只需要 50 个循环,但我们通常订购 75 个循环并在数据分析步骤期间截断读数。
b. 指数 I7:7 个周期
c. 测序深度:1M 读数/样本。
d. 读取 1 的定制测序引物:T7-Illumina-READ1-A


数据分析


笔记:
a. 在下面的说明中,代码行以粗体显示。这些说明适用于使用 SLURM 的计算集群。
b. 提供了两个血清样本及其技术复制的示例VirScan数据(补充材料)。数据文件包括样本图例、 fastq文件、BAM 文件、对齐报告文件、索引 BAM 文件、计数文件、 count.combined文件( Nextseq 500 流动槽的四个通道的计数总和)、计数表( count.combined数据以表格形式呈现;无血清对照的总计数在“输入”列中提供),Z 分数表(同样,无血清对照的总计数出现在“输入”列中), hits_combined表和病毒分数文件。


A. 将测序读数与参考文件对齐
1. VirScan库的参考fasta文件(“vir3.fasta”)(补充材料)并生成带有 . ebwt扩展。运行以下命令:
模块加载gcc /6.2.0


模块加载蝴蝶结/1.2.2


领结构建 vir3.fasta vir3
2. 将测序读数与参考文件对齐。请参阅“script.align.sh”并根据需要进行编辑(补充材料)。输出文件是以“.bam ”结尾的文件
笔记:
a. 测序读取通常以fastq文件的形式分发。这些fastq文件存储在名为“ raw.data ”的子目录中。
b. 在“script.align.sh”中,“bowtie -3 25”从每个测序读数的 3' 端修剪 25 个核苷酸。如果测序读数长度为 75 个核苷酸,则执行此操作。参考文件仅包括文库每个成员的前 50 个核苷酸,因此必须将测序读数缩减至 50 个核苷酸才能与参考正确对齐。
c. 在“script.align.sh”中,将“path_to_vir3_reference_fasta_and_index_files”替换为适当的路径。
./ script.align.sh
3. “.out”结尾的对齐报告文件
注意:通常,>85% 的读取与参考文件对齐。
4. 使用以下命令索引文件。输出文件是以“.bai ”结尾的文件
模块加载gcc /6.2.0


模块加载samtools /1.3.1


对于我在raw.data /*.bam;做samtools索引 $ i ;完毕
5. 使用以下命令计算索引。输出是一个以“.count.csv ”结尾的文件
模块加载gcc /6.2.0


模块加载samtools /1.3.1


对于我在raw.data /*.bam;做samtools idxstats $ i |切-f 1,3 | sed -e '/^\*\t/d' -e '1 i id\ tSAMPLE_ID ' | tr "\\t" "," >${ i %.bam }.count.csv ;完毕
6. 压缩包 使用以下命令计数文件。
对于我在raw.data /*.csv;做gzip $ i ;完毕
7. 使用以下命令创建一个名为“ log_directory ”的目录。
mkdir 日志目录
8. 如果 同一样品在流动槽的两个或多个通道上运行,并且为每个流动槽提供单独的文件,请使用以下命令组合来自不同通道的计数文件。这些命令需要将 python 脚本“ combine_two_lanes.py”复制到运行命令的文件夹(补充材料)。
注意:在下面的代码中,样本在 Illumina Nextseq 500 流动槽的四个通道上运行。每个计数文件的后缀是“L001_R1_001.count.csv.gz”,如果计数文件来自流动槽的第一道,“L002_R1_001.count.csv.gz”如果计数来自流动的第二道细胞等
模块加载gcc /6.2.0


模块加载python


对于我在raw.data /*L001_R1_001.count.csv.gz;做python combine_two_lanes.py $ i $ { i%1_R1_001.count.csv.gz } 2_R1_001.count.csv.gz $ {i%1_R1_001.count.csv.gz } 1_2_R1_001.count.csv ;完毕


对于我在raw.data /*L003_R1_001.count.csv.gz;做python combine_two_lanes.py $ i $ { i%3_R1_001.count.csv.gz } 4_R1_001.count.csv.gz $ {i%3_R1_001.count.csv.gz } 3_4_R1_001.count.csv;完毕


对于我在raw.data /*L001_2_R1_001.count.csv;做python combine_two_lanes.py $ i $ { i%1_2_R1_001.count.csv } 3_4_R1_001.count.csv $ {i%1_2_R1_001.count.csv } 1_2_3_4_R1_001.count.combined.csv ;完毕


9. 压缩包 count.combined文件使用以下命令。
对于我在raw.data /*1_2_3_4_R1.count.combined.csv;做gzip $ i ;完毕


B. 计算 Z 分数
注意:为了执行 Z 分数分析,将 count.combined文件合并到一个表中,并将与无血清对照对应的列汇总在一个称为“输入”的列中。
1. 编辑 R 脚本“ Zscore_analysis.R ”以包含count.combined表文件的路径和输出文件的所需路径,然后运行脚本(补充材料)。需要包“mmR_0.1.0”和“virScanR_0.1.0.9000”(补充材料)。
注意:文件“Zscores_vir3”包含此步骤后的结果(补充材料)。
2. 在样品的两个技术重复中,Z 分数至少为 3.5 才能将肽称为“命中”。
注意:文件“hits_combined_vir3_3.5_cutoff”包含此步骤之后的结果(补充材料)。


C. 计算病毒分数
1. 创建一个名为“hits”的目录。在这个目录中应该是每个样本的 .csv 文件,每个肽段 ID 的值为“True”或“False”,具体取决于肽段在样本的两个技术复制中是否得分为命中(Z 得分 > 3.5) .这些文件可以通过将“ hits_combined_vir3_3.5_cutoff”文件的每一列拆分为单独的文件(补充材料)来创建。
2. 使用以下代码生成病毒分数文件:
注意: “VIR3_clean”文件提供了寡核苷酸的注释”(补充材料) 。这里是 Vir 3 库中的 115,753 个寡核苷酸。一些蛋白质片段在不同病毒中是相同的,在这种情况下,“VIR3_clean”文件中有多行对应于一个寡核苷酸。要识别给定肽的病毒来源,请在 VIR3_clean 文件中查找具有给定肽“id”值的行。 


对于我在 hits/*.csv.gz 中;做 python calc_scores_nofilter.py $ i VIR3_clean.csv.gz 物种 7 >virus_scores_$ i ;完毕


D. 确定病毒血清阳性
1. 如果virus_score > VirScan_viral_threshold并且如果来自该病毒的至少一个公共表位得分为命中,则确定样品对病毒血清阳性。 “ VirScan_viral_thresholds ”文件包含每个病毒的阈值(补充材料)。
注意:可根据要求提供公共表位注释。


食谱


1. 噬菌体提取缓冲液
20 mM Tris-HCl,pH 8.0
100 毫米氯化钠
6 毫米硫酸镁4
储存于 4°C


2. PhIP -Seq 洗涤缓冲液
50 mM Tris-HCl,pH 7.5
150 毫米氯化钠
0.1% NP-40
储存于 4°C


致谢


有趣的:ELS 得到了 NSF 研究生研究奖学金计划的支持。 SJE 是霍华德休斯医学研究所的研究员。
源自该协议的原始研究论文: Larman 等。 (2011),徐等人。 (2015)和米娜等人。 (2019)。
我们感谢 A. Kohlgruber在图形摘要中设计示意图。


利益争夺


SJE 是 TSCAN Therapeutics、MAZE Therapeutics、 Mirimus和ImmuneID的创始人。 SJE 是 Homology Medicines、TSCAN Therapeutics、MAZE Therapeutics、 XChem的科学顾问委员会成员,并且是 MPM 的顾问,这些都不会影响这项工作。 ELS曾担任ImmuneID的顾问。 SJE 是布莱根妇女医院 (US20160320406A) 提交的专利申请的发明人,该专利申请涵盖了使用VirScan库来识别血液中的病原体抗体。


伦理


在获得捐赠者的书面知情同意后,根据当地管理人类研究的协议收集人体样本。布莱根妇女医院机构审查委员会(协议号 2013P001337)豁免了出于这项工作的目的对所有人体样本的二次使用。


参考


1. Bjornevik , K., Cortese, M., Healy, BC, Kuhle , J., Mina, MJ, Leng , Y., Elledge , SJ, Niebuhr, DW, Scher , AI, Munger, KL等。 (2022 年)。纵向分析显示与多发性硬化症相关的 Epstein-Barr 病毒的高流行率。 科学375(6578):296-301。
2. Borriello , F., Poli , V., Shrock , E., Spreafico , R., Liu, X., Pishesha , N., Carpenet , C., Chou, J., Di Gioia , M., McGrath, ME,等人。 (2022 年)。通过调节微生物配体的物理特性实现的佐剂策略扩大了抗原免疫原性。 单元格185(4):614-629 e621。
3. Chen, G., Shrock , EL, Li, MZ, Spergel , JM, Nadeau, KC, Pongracic , JA, Umetsu , DT, Rachid, R., MacGinnitie , AJ, Phipatanakul , W.等。 (2021 年)。 AllerScan 的高分辨率表位作图揭示了花生口服免疫治疗期间 IgE 和 IgG 库之间的关系。 细胞代表医学2(10):100410。
4. Garrett, ME, Itell , HL, Crawford, KHD, Basom , R., Bloom, JD 和Overbaugh , J. (2020)。噬菌体-DMS:抗体表位精细定位的综合方法。 iScience 23(10):101622。
5. Hill, JA, Krantz, EM, Hay, KA, Dasgupta, S., Stevens-Ayers, T., Bender Ignacio, RA, Bar, M., Maalouf , J., Cherian, S., Chen, X.等. (2019)。 CD19 定向嵌合抗原受体 T 细胞免疫治疗后抗病毒抗体的持久保存。 血液 Adv 3(22): 3590-3601。
6. Kosuri , S., Eroshenko , N., Leproust , EM, Super, M., Way, J., Li, JB 和 Church, GM (2010)。通过选择性扩增来自高保真微芯片的 DNA 库进行可扩展的基因合成。 Nat Biotechnol 28(12):1295-1299。
7. Langmead, B.、 Trapnell , C.、Pop, M. 和Salzberg , SL (2009)。短 DNA 序列与人类基因组的超快和高效记忆比对。 基因组生物学10(3):R25。
8. Larman ,HB,Zhao,Z., Laserson ,U.,Li,MZ, Ciccia ,A., Gakidis ,MA,Church,GM, Kesari ,S., Leproust ,EM, Solimini ,NL等。 (2011)。使用合成人类肽组发现自身抗原。 Nat Biotechnol 29(6):535-541。
9. Li, H., Handsaker , B., Wysoker , A., Fennell, T., Ruan , J., Homer, N., Marth, G., Abecasis , G., Durbin, R. and Genome Project Data Processing, S. (2009)。序列比对/映射格式和 SAMtools。 生物信息学25(16):2078-2079。
10. Mandel-Brehm, C., Dubey, D., Kryzer , TJ, O'Donovan, BD, Tran, B., Vazquez, SE, Sample, HA, Zorn, KC, Khan, LM, Bledsoe, IO等。 (2019)。精原细胞瘤相关副肿瘤性脑炎中的 Kelch 样蛋白 11 抗体。 N Engl J Med 381(1):47-54。
11. Mina,MJ,Kula,T., Leng ,Y.,Li,M.,de Vries,RD, Knip ,M., Siljander ,H., Rewers ,M.,Choy,DF,Wilson,MS等。 (2019)。麻疹病毒感染会减少预先存在的抗体,这些抗体可以提供对其他病原体的保护。 科学366(6465):599-606。
12. Mohan, D., Wansley, DL, Sie, BM, Noon, MS, Baer, AN, Laserson , U. 和Larman , HB (2018)。使用寡核苷酸编码的肽组对血清抗体进行 PhIP-Seq 表征。 国家协议13(9):1958-1978 。
13. Pou , C., Nkulikiyimfura , D., Henckel , E., Olin, A., Lakshmikanth , T., Mikes, J., Wang, J., Chen, Y., Bernhardsson , AK, Gustafsson, A.等人_ (2019)。人类新生儿中的母体抗病毒抗体库。 Nat Med 25(4):591-596。
14. R 核心团队。 (2017)。 R:统计计算的语言和环境。 R 统计计算基金会,奥地利维也纳。 ” https://www.r-project.org/ ”。
15. Schubert, RD, Hawes, IA, Ramachandran, PS, Ramesh, A., Crawford, ED, Pak, JE, Wu, W., Cheung, CK, O'Donovan, BD, Tato, CM等。 (2019)。泛病毒血清学表明肠道病毒与急性弛缓性脊髓炎有关。 Nat Med 25(11):1748-1752。
16. Shrock,E.,Fujimura,E.,Kula,T.,Timms,RT,Lee,IH, Leng ,Y.,Robinson,ML,Sie,BM,Li,MZ,Chen,Y.,等。 (2020 年)。 COVID-19 患者的病毒表位分析揭示了交叉反应性和严重程度的相关性。 科学370(6520)。
17. Smith, GP 和Petrenko , VA (1997)。噬菌体展示。 化学修订版 97(2):391-410。
18. Xu, GJ, Kula, T., Xu, Q., Li, MZ, Vernon, SD, Ndung'u , T., Ruxrungtham , K., Sanchez, J., Brander, C., Chung, RT, et al . (2015 年)。病毒免疫学。使用合成人类病毒组对人群进行综合血清学分析。 科学348(6239):aaa0698。
19. Zamecnik , CR, Rajan , JV, Yamauchi, KA, Mann, SA, Loudermilk, RP, Sowa, GM, Zorn, KC, Alvarenga, BD, Gaebler , C., Caskey, M.等。 (2020 年)。 ReScan 是一种多重诊断管道,可检测 SARS-CoV-2 抗原的人血清。 细胞代表医学1(7):100123。


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引用:Shrock, E. L., Shrock, C. L. and Elledge, S. J. (2022). VirScan: High-throughput Profiling of Antiviral Antibody Epitopes . Bio-protocol 12(13): e4464. DOI: 10.21769/BioProtoc.4464.
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