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Apr 2020
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Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome
用于分析冠状病毒基因组中广谱抗病毒药物诱导的过渡突变和错误率的引物ID下一代测序   

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Abstract

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.

Keywords: Coronavirus (冠状病毒), Antivirals (抗病毒药), Next generation sequencing (新一代测序), Primer ID (引物 ID), Mutation (突变)

Background

Next generation sequencing (NGS) has been extensively used in biomedical research for the last decade. When applying NGS to study intra-host viral populations for RNA viruses, modifications in library prep and sequencing protocols need to be considered. The virus titers (or viral loads) vary greatly from specimen to specimen. While conventional NGS platforms require 1-500 ng of DNA (or RNA) in a sequencing run, in most cases a clinical sample will have less than 100 fg of viral RNA, requiring that the viral RNA first be converted to cDNA using reverse transcriptase, followed by one or two rounds of PCR amplification to generate enough material for sequencing. However, the extensive cycles of PCR amplification will cause nucleotide mis-incorporation, recombination and amplification bias. Furthermore, the NGS platforms have relatively high error rates adding additional uncertainty to the resulting sequences (Liu et al., 2012).


We have developed the Primer ID NGS approach to overcome the errors and bias from the conventional NGS approaches (Jabara et al., 2011; Zhou et al., 2015). During the initial cDNA synthesis step, we use cDNA primers with an embedded 11-base degenerate block of nucleotides (the Primer ID) to tag each viral RNA template with a unique ID, a version of the approach of adding a unique molecular identifier (UMI). The Primer ID is carried on throughout the downstream amplifications and sequencing. After sequencing, all raw sequences with the same ID are collapsed to make a template consensus sequence (TCS). Each TCS is linked to an original template that was queried in the initial cDNA synthesis step, and the number of TCS shows the sequencing sampling depth of the viral population. By the construction of consensus sequence from multiple raw sequencing reads for each viral template, we can greatly reduce the PCR and sequencing errors. We have shown that the Primer ID approach can greatly reduce the sequencing error to 1 in 10,000 nucleotides (100-fold reduction from the raw sequencing reads) and also reveal the true sampling depth of the viral population, making it possible to accurately measure the substitution rates and diversity in the viral genomes in a population (Figure 1) (Zhou et al., 2015). We have further developed a multiplexing Primer ID approach, allowing the sequencing of multiple regions of the viral genome in a single cDNA synthesis/PCR (Figure 2) (Dennis et al., 2018).



Figure 1. Primer ID Approach. A. The structure of the Primer ID primer and its binding to the viral RNA template. B. An example of creating the template consensus sequence from raw sequence reads.



Figure 2. Library prep steps for the Multiplexed Primer ID sequencing. Biological sequence region in blue color, Primer ID region in yellow color, forward spacer region in orange, MiSeq barcode sequence region in purple.


This approach is ideal for studying intra-host RNA virus populations due to its ability to accurately characterize individual viral genomes. We have used this approach to determine the genetic structures of intra-host viral populations in HIV (Zhou et al., 2016; Lee et al., 2017; Dennis et al., 2018; Abrahams et al., 2019; Adewumi et al., 2020; Council et al., 2020), to study drug resistance mutations in HIV and HCV (Clutter et al., 2017; Zhou et al., 2018; Keys et al., 2015), and to detect nucleotide mutation rates in single-stranded DNA molecule after heating (Lewis et al., 2016).


In this report we describe a protocol using the Primer ID MiSeq sequencing to detect the mutagenic effect of EIDD-1931(β-D-N4-hydroxycytidine, NHC) and EIDD-2801 (an orally bioavailable prodrug of EIDD-1931 and is also recognized as MK-4482 or Molnupiravir) in the Middle East respiratory syndrome coronavirus (MERS-CoV) genome from in vitro cell culture and in vivo mouse models. In the cell culture sequencing experiment, we used a multiplexed Primer ID protocol to sequence multiple regions in the MERS-CoV ORF1b in a single library prep and sequencing reaction, while in the sequencing of total lung RNA from MERS-CoV infected mice, we used a multiplexed Primer ID protocol targeting both MERS-CoV genomes and two selected mouse mRNAs in a single library prep and sequencing reaction, in which we can directly compared the mutagenic effect induced by EIDD-2801 on MERS-CoV genomes and host mRNA. This protocol is not restricted to MERS-CoV and EIDD-1931 study only, but it can be used to detect viral RNA mutation rate in general.

Materials and Reagents

  1. Pipette tips

  2. Collection tubes (QIAGEN, catalog number: 19201, stored at room temperature)

  3. Qubit Assay Tubes (ThermoFisher Scientific, Invitrogen, catalog number: Q32856, stored at room temperature)

  4. SuperScript III One-Step RT-PCR System (ThermoFisher Scientific, Invitrogen, catalog number: 12574026, stored at -20 °C)

  5. Primer ID primers (Integrated DNA Technologies) at 100 µM in Tris-HCl buffer stored at -20 °C

  6. SuperScript III Reverse Transcriptase (ThermoFisher Scientific, Invitrogen, catalog number: 18080085, stored at -20 °C)

  7. RNaseOUT Recombinant Ribonuclease Inhibitor (ThermoFisher Scientific, Invitrogen, catalog number: 10777019, stored at -20 °C)

  8. Ribonuclease H (ThermoFisher Scientific, Invitrogen, catalog number: 18021071, stored at -20 °C)

  9. KAPA2G Robust PCR kits (Hotstart PCR kits with dNTPs) (Roche, catalog number: KK5516, stored at -20 °C)

  10. KAPA2G HiFi HotStart (with dNTPs) (Roche, catalog number: KK2502, stored at -20 °C)

  11. RNAClean XP (Beckman Coulter, catalog number: A63987, stored at 4 °C)

  12. AMPure XP (Beckman Coulter, catalog number: A63881, stored at 4 °C)

  13. Ethanol 200 Proof (Decon Laboratories, catalog number: 2716)

  14. DNase/RNase-free water

  15. MinElute Gel Extraction kit (QIAGEN, catalog number: 28606, stored at 4 °C for MinElute columns, room temperature for other reagents)

  16. Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Invitrogen, catalog number: Q32853, stored at 4 °C for standard 1 and standard 2, room temperature for other reagents)

  17. Experion DNA 12K analysis kit (Bio-Rad, catalog number: 7007108, stored at room temperature for the chips, 4 °C for other reagents)

  18. MiSeq Reagent Kit v3 (600-cycle) (Illumina, catalog number: MS-102-3003, stored at -20 °C for box 1 and 4 °C for box 2)

  19. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465, stored at room temperature)

  20. Tris hydrochloride (Sigma-Aldrich, catalog number: 10812846001, stored at room temperature)

  21. PhiX Control V3 (Illumina, catalog number: FC-110-3001, stored at -20 °C)

  22. 10 mM Tris-HCl with 0.1% TWEEN 20 (Teknova, catalog number: T7724, stored at room temperature)

Equipment

  1. Pipettes

  2. Magnetic rack

  3. Thermal cycler (ThermoFisher Scientific, Applied Biosystems, catalog number: 4384638)

  4. Centrifuge (Eppendorf, model: 5424)

  5. DynaMag-2 Magnet (ThermoFisher Scientific, Invitrogen, catalog number: 12321D)

  6. Qubit 2.0 Fluorometer (ThermoFisher Scientific, Invitrogen, catalog number: Q32866)

  7. Experion Electrophoresis Station (Bio-Rad, catalog number: 7007010)

  8. MiSeq System (Illumina, catalog number: SY-410-1003)

Software

  1. Primer-BLAST (NCBI, https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Ye et al., 2012)

  2. bcl2fastq pipeline (v.2.20.0) (Illumina, https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html)

  3. TCS web app (UNC, https://tcs-dr-dept-tcs.cloudapps.unc.edu/)

  4. TCS pipeline (v.1.3.8) (UNC, https://github.com/SwanstromLab/PID)

  5. RUBY package viral_seq (v.1.0.8) (UNC, https://github.com/ViralSeq/viral_seq)

Procedure

We first design the primers for the multiplexed Primer ID library prep. After extraction of viral RNA from the tissue culture supernatants or total RNA from lung tissues, we construct MiSeq sequencing libraries using the multiplexed Primer ID library prep protocol from each specimen. The multiplexed Primer ID protocol allows us to make a sequencing library of multiple regions of the viral RNA in one reaction. We pool up to 24 barcoded libraries (from 24 different specimens) for each sequencing run. We use Illumina MiSeq V3 kit (600 cycle) for the sequencing. We choose paired-end sequencing to have longer read length and better error correction from overlapping regions over single-end sequencing.


  1. Primer ID cDNA primer and PCR primer design

    1. Design primer sequences using NCBI Primer-BLAST.

      1. Use ORF1b region of the reference genome MERS-CoV NC_019843 as “PCR template” in the Primer-BLAST interface to design primers for MERS-CoV. Use mouse interferon-induced protein with tetratricopeptide repeats 2 (Ifit2, reference: NM_008332), interferon-induced protein with tetratricopeptide repeats 3 (Ifit3, reference: NM_010501), ISG15 ubiquitin-like modifier (Isg15, reference: NM_015783), interferon-induced protein with tetratricopeptide repeats 1 (Ifit1, reference: NM_008331) and chemokine (C-X-C motif) ligand 10 (Cxcl10, reference: NM_021274) genomes as templates to design primers for mouse mRNA.

      2. Set PCR product size as Min: 400 bp and Max: 600 bp.

      3. Set Primer melting temperatures as Min: 52, Opt: 55, Max: 58.

      4. Select 3-4 pairs of primers for each template region.

    2. Use SuperScript III one-step RT-PCR system to test Primers with best amplification efficiency for each region.

      1. Use MERS-CoV viral RNA from tissue culture described above for MERS-CoV primer testing. Use extracted total mouse lung RNA as template for mouse mRNA primer testing. Make serial 1:10 titrations of the template RNA in DNase/RNase-free water as RT-PCR templates. Make dilutions up to 10-6.

      2. Use following recipe to make one one-step RT-PCR mixture (total volume of 10 µl) in PCR tubes.

        5 µl of 2× buffer

        1.8 µl of DNase/RNase-free water

        0.4 µl of Forward primer (20 µM)

        0.4 µl of Reverse R (20 µM)

        0.4 µl of Enzyme mix

        2 µl of Template RNA

      3. Use the following thermal cycler condition for RT-PCR.

        1. 50 °C for 30 min

        2. 94 °C for 2 min

        3. 40 cycles of:

          94 °C for 15 s

          55 °C for 30 s

          68 °C for 30 s

        4. 68 °C for 5 min

      4. Examine the PCR products using gel electrophoresis with 1% agarose gel.

      5. Figure 3 shows the example gel image of amplicons from tested primers for MERS-CoV.



      Figure 3. An example one-step RT-PCR gel image for the primer design. Three sets of primers targeting same MERS-CoV genome were tested. A serial dilution of MERS-CoV RNA up to 10-6 dilution was used to test the primers. The SSIII one-step PCR system was used for the amplification. PCR products were examined by gel electrophoresis with 1% agarose gel. Primer set 2 had the best amplification efficiency thus it was selected for downstream Primer ID primer design. NC, no-reaction control.


    3. Design the Primer ID cDNA primers and other primers for the MiSeq library prep.

      1. Design cDNA primers by attaching the Primer ID (a degenerate block of 11-base long sequence) and adaptor sequence to the reverse primers.

      2. Forward primer of the 1st round PCR is designed by joining a common sequence “GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNN” on the 5’ end with the forward primer sequencing designed at step a) on the 3’. The Ns in the joined primer sequences are random sequences to increase the sequencing quality.

      3. Reverse primer of the 1st round PCR is designed against the adaptor sequence section of the Primer ID cDNA primer, and is universal regardless of regions (Table 1).

      4. The 2nd round PCR primers are designed to incorporate the MiSeq sequencing adaptors with barcodes.

      5. Order primers designed above in Steps A1a-A1d from Integrated DNA Technologies (IDT). Request hand-mixing of the precursors for the degenerate block of sequence of the cDNA primers to have an even distribution of bases at each position.

      6. Table 1 shows the complete list of primers used in this protocol including primers for Primer ID MiSeq library prep and customized MiSeq sequencing primer. Table 2 shows the sequences of the indexed primers.

    Notes:

    1. Primer-BLAST is not necessary to design primers. Primers can be manually designed based on known reference genomes.

    2. Hand-mixing of nucleotide precursors prior to Primer ID cDNA primer synthesis is important to ensure the degenerate sequences have an even distribution of bases (A, C, G, T) at each position of the degenerate block.

      Table 1. Primers used for MiSeq library prep and sequencing. Primer sequences with gene-specific regions are highlighted in blue. They can be replaced with other gene-specific primer sequences when designing primers for other viruses.

      Primer 5'-3' sequence Comment
      41R_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTATGACCTTCCTGTTGCTTCT cDNA primer. Targeting 20331-20350 on the reference genome.
      nsp10_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCCTAAAGACGACATCAGTGG cDNA primer. Targeting 13488-13507 on the reference genome.
      nsp12_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTATAGCCAAAGACACAAACCG cDNA primer. Targeting 15983-16002 on the reference genome.
      nsp14_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTGAACATCGACAAAGAAAGGG cDNA primer. Targeting 18715-18734 on the reference genome.
      ifit3_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTTTCAGCCACTCCTTTATCCC cDNA primer. Targeting mice IFIT3 mRNA.
      isg15_PID11* GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTGGGGCTTTAGGCCATACTC cDNA primer. Targeting mice ISG15 mRNA.
      41F_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNGCTACAAGTTCGTCCTTTGG 1st round PCR forward primer. Targeting 19812-19831 on the reference genome
      nsp10_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTGCTCAGGTGCTAAGCGAAT 1st round PCR forward primer. Targeting 12983-13002 on the reference genome
      nsp12_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNATAGGCTTCGATGTTGAGGG 1st round PCR forward primer. Targeting 15388-15407 on the reference genome
      nsp14_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNATTGCAAGCTGGTTCTAACA 1st round PCR forward primer. Targeting 18260-18279 on the reference genome
      ifit3_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNCGATCCACAGTGAACAACAG 1st round PCR forward primer. Targeting mice IFIT3 mRNA.
      isg15_AD GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTGGGACCTAAAGGTGAAGATG 1st round PCR forward primer. Targeting mice ISG15 mRNA.
      Adapter R GTGACTGGAGTTCAGACGTGTGCTC 1st round PCR reverse primer
      Universal Adapter AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTG 2nd round PCR forward primer with Illumina adapter sequence
      Indexed Adapter** CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTC 2nd round PCR reverse primer with Illumina adapter sequence and indices (NNNNNNN)
      Old Nextera GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG Customized sequencing primer


      * cDNA primer has a block of degenerate nucleotides (Ns) as the Primer ID.
      ** Ns in the Indexed Adapter primers are a set of 24 pre-designed barcodes for multiplexing samples for one MiSeq run. See the index sequences and adapter sequences in Table 2.

      Table 2. Indexed adapter sequences. Note the index sequences are the reverse complement sequences of the highlighted (red) region in the adapter sequences
      Indexed Adapter Index Index Sequence Sequence (5’-3’)
      PCR Primer, Index 1 1 ATCACGA CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 2 2 CGATGTA CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 3 3 TTAGGCA CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 4 4 TGACCAA CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 5 5 ACAGTGA CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 6 6 GCCAATA CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 7 7 CAGATCA CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 8 8 ACTTGAA CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 9 9 GATCAGA CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 10 10 TAGCTTA CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 11 11 GGCTACA CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 12 12 CTTGTAA CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 13 13 TCCATAA CAAGCAGAAGACGGCATACGAGATTATGGAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 14 14 GTACTAA CAAGCAGAAGACGGCATACGAGATTAGTACGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 15 15 ACAGTAA CAAGCAGAAGACGGCATACGAGATTACTGTGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 16 16 CTCATGA CAAGCAGAAGACGGCATACGAGATCATGAGGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 17 17 ACGATAA CAAGCAGAAGACGGCATACGAGATTATCGTGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 18 18 TGCAGAA CAAGCAGAAGACGGCATACGAGATTCTGCAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 19 19 TTCATAA CAAGCAGAAGACGGCATACGAGATTATGAAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 20 20 TGCTGTA CAAGCAGAAGACGGCATACGAGATACAGCAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 21 21 TATCACA CAAGCAGAAGACGGCATACGAGATGTGATAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 22 22 TGGATAA CAAGCAGAAGACGGCATACGAGATTATCCAGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 23 23 CGCATTA CAAGCAGAAGACGGCATACGAGATAATGCGGTGACTGGAGTTCAGACGTGTGCTC
      PCR Primer, Index 24 24 GCCTTAA CAAGCAGAAGACGGCATACGAGATTAAGGCGTGACTGGAGTTCAGACGTGTGCTC

  2. Primer ID MiSeq library prep

    1. cDNA synthesis

      1. Use viral RNA extracted from cell culture supernatants from infected cells or total lung tissue RNA from infected mice as amplification templates. cDNA synthesis step should be performed in BSL2+ facility until after the Ribonuclease H step.

      2. Completely thaw, vortex and spin down 5× buffer, dNTPs and DTT.

      3. Make cDNA primer working mix from the 100 µM primer stock. The final concentration for each primer is 10 µM in the primer mix.

        1. Determine regions for sequencing.

        2. Add 10 µl of each cDNA primer (no more than 5 different primers to multiplex) to an Eppendorf microcentrifuge tube, and add DNase/RNase-free water to a total volume of 100 µl.

        3. Increase the amount of primers and DNase/RNase-free water proportionally if larger volume of primer mix is needed.

      4. Pipette the following components into a 0.5 ml RNase-free PCR tube.

        2 µl of dNTP mix (10 mM each)

        1 µl of Primer ID cDNA primer mix (10 µM each)

        23 µl of RNA template (vRNA or total lung RNA)

      5. Heat the PCR tube on a thermal cycler at 65°C for 5 min, then cool down to 4 °C for 2 min with lid closed (lid temperature at 105 °C).

      6. Add the following components to the tube. If doing the cDNA synthesis in batch, make a master mix and add 14 µl of the mixture to each tube.

        8 µl of 5× buffer

        2 µl of DTT (100 mM)

        2 µl of RNaseOUT (40 U/µl)

        2 µl of SSIII reverse transcriptase (200 U/µl)

      7. Mix well by pipetting up and down for a few times. Transfer the tube(s) to a thermal cycler. Use the following thermal cycler program for the cDNA synthesis.

        55 °C for 1 h

        50 °C for 1 h

        70 °C for 15 min

      8. To each tube, add 0.5 μl (1U) Ribonuclease H, incubate at 37 °C for 20 min. After this step, the cDNA can be handled on a regular workbench (BSL1).

      9. Purify cDNA with RNAClean XP.

        1. Resuspend the beads by vortexing and take an aliquot out. Keep in room temperature for at least 30 min before use.

        2. Resuspend the beads thoroughly by vortexing. Add 28 µl of RNAClean XP beads to 1.5 ml tubes. Transfer all of the cDNA reaction (40.5 µl) to each tubes with beads.

        3. Mix the RNAClean XP and sample thoroughly by pipette mixing 15 times. Vortexing is not recommended. Let the tube incubate at room temperature for 20 min. Spin down briefly if samples touches the upper part of the tube.

        4. Place the tube onto the magnetic tube rack for 5 min to separate the beads from solution.

        5. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not disturb the magnetic beads, which have formed a spot on the side of the tube.

        6. Dispense 400 μl of 70% ethanol into the tube and incubate for 30 s at room temperature. Aspirate out the ethanol and discard. Repeat for a total of three washes. It is important to perform these steps with the tube situated on the magnetic rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.

        7. Let the reaction tube air-dry up to 10 min on the rack with the cap open. The tube(s) should air-dry until the last visible traces of ethanol evaporate. However, over drying the sample may result in a lower recovery.

        8. Remove tube from magnetic rack and resuspend beads in 24 µl DNase/RNase-free water by pipetting up and down. Place tube back on rack and leave for 3 min.

        9. Pipette the eluent (23.5 µl) from the tube while it is situated on the magnetic tube rack.

    2. First round PCR amplification with KAPA2G Robust PCR kit.

      In the first round PCR, we amplify all the cDNA we make from the previous step using a mixture of forward primers targeting multiple regions of the genome.

      1. Make forward primer working mix from the 100 µM primer stock.

        1. Determine regions of primer binding based on the initial choice of placement of the cDNA primers.

        2. Add 10 µl of each forward primer (no more than 5 primers) to an Eppendorf microcentrifuge tube, and add DNase/RNase-free water to a total volume of 100 µl.

        3. Increase the amount of primers and DNase/RNase-free water proportionally if larger volume of primer mix is needed.

      2. Completely thaw, vortex and spin down reagents (except for enzyme) before use.

      3. Use the following recipe to make one first round PCR mix (total volume of 50 µl).

        10 µl of 5× Buffer A

        10 µl of Enhancer

        1 µl of dNTPs

        2.5 µl of Forward primer mix

        2.5 µl of adapter R primer

        0.5 µl of KAPA Robust polymerase

        23.5 µl of template cDNA

      4. Mix well by pipetting up and down for a few times. Transfer the tube(s) to a thermal cycler. Use the following thermal cycler program for the first round PCR amplification.

        1. 95 °C for 1 min

        2. 25 cycles of:

          95 °C for 15 s

          58 °C for 60 s

          72 °C for 30 s

        3. 72 °C for 3 min

      5. Purify PCR products using AMPure XP cleanup beads.

        1. Vortex the 1 ml aliquot of AMPure XP cleanup beads and remove the needed volume. Keep in room temperature for at least 30 min before use.

        2. Transfer the first round of PCR reactions into 1.5 ml DNase-free tubes.

        3. Resuspend the beads. Add 35 µl AMPure XP beads to each PCR reaction.

        4. Mix the AMPure XP and sample thoroughly by vortexing. Let the tube incubate at room temperature for 5 min before proceeding to the next step (incubate off the rack).

        5. Place the tube onto the magnetic tube rack for 5 min to separate the beads from solution.

        6. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not disturb the magnetic beads, which have formed a spot on the side of the tube.

        7. Dispense 400 μl of 70% ethanol into the tube and incubate for 30 s at room temperature. Aspirate out the ethanol and discard. Repeat for a total of two washes. It is important to perform these steps with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.

        8. Let the reaction tube air-dry up to 10 min on the rack with the cap open. The tube(s) should air-dry until the last visible traces of ethanol evaporate. Over drying the sample may result in a lower recovery.

        9. Remove tube from rack and resuspend beads in 50 µl DNase-free water by pipetting up and down. Place tube back on rack and leave for 3 min.

        10. Pipette the 45 µl eluent from the tube while it is situated on the magnetic tube rack. Transfer the eluent to a new 1.5 ml microcentrifuge tube.

    3. Second round of PCR amplification with KAPA2G HiFi PCR kits.

      1. Complete thaw, vortex and spin down reagents (except for enzyme) before use.

      2. Use the following recipe to make one second round PCR mix (total volume of 25 µl).

        10 µl of 5× KAPA HiFi Buffer

        1 µl of dNTPs

        1 µl of Universal Adapter

        1 µl of indexed Adapter

        0.5 µl of KAPA HiFi polymerase

        2 µl of purified DNA from the first round PCR.

      3. Mix well by pipetting up and down for a few times. Transfer the tube(s) to a thermal cycler. Use the following thermal cycler program for the cDNA synthesis.

        1. 95 °C for 2 min

        2. 35 cycles of:

          98 °C for 20 s

          63 °C for 15 s

          72 °C for 30 s

        3. 72 °C for 3 min

    4. Gel purification of PCR products using QIAGEN MinElute Gel extraction kit.

      1. Before gel extraction, run 2 µl PCR products on 1% agarose gel to check if the library has the right size.

      2. Make 1.2% (w/v) agarose gel with ethidium bromide (final concentration of 0.2-0.5 µg/ml).

      3. Load all the second round PCR products on the 1.2% agarose gel. Adjust the voltage based on the size of the gel. The ideal electric field strength is 4 V/cm. Run the gel for 30 min.

      4. Excise target DNA fragments of the right size (500 bp to 1 kb) on a non-UV blue light transilluminator.

      5. Weigh the gel; add 3 volumes of Buffer QG to 1 volume of gel.

      6. Incubate at 50 °C for 10 min to completely dissolve the gel fragment. Vortex every 2-3 min to help dissolve.

      7. Check the color of the gel solution (should be yellow, otherwise add 10 µl 3 M sodium acetate of pH 5.0).

      8. Apply the sample to the MinElute column and centrifuge at maximum speed for 1 min.

      9. Transfer the column to a new collection tube, discard the filtrate, add 500 µl buffer QG and centrifuge at maximum speed for 1 min.

      10. Transfer the column to a new collection tube, discard the filtrate, add 0.75 ml buffer PE, incubate for 5 min at room temperature, centrifuge at maximum speed for 1 min.

      11. Transfer the column to a new collection tube, discard the filtrate, centrifuge at maximum speed for additional 3 min.

      12. Put the column in a new 1.5 ml tube, add 10 µl buffer EB. Stand for 4 min, centrifuge at maximum speed for 2 min.

    5. Quantification, pooling and QA/QC.

      1. Quantify each gel-purified library using Qubit dsDNA BR Assay kit, following the manufacturer’s protocol. Use 1 µl of the library for each quantification.

      2. Pool the quantified individual libraries in equal amounts. We suggest pooling a total number of 20-24 libraries, each with 100 to 150 ng DNA per library. Use the maximum available volume of libraries if the calculated volume exceeds the actual volume.

      3. Purify the pool of libraries with AMPure XP beads following the protocol above in the First Round PCR section. The volume of AMPure beads to use equals the volume of pooled libraries multiplied by 0.7.

      4. Quantify the purified pooled libraries using Qubit dsDNA BR Assay kit, following the manufacturer’s protocol.

      5. Run the pooled libraries with the Experion Automated Electrophoresis System with DNA 12k analysis kit following the manufacturer’s protocol. An example of gel image of Experion is shown in Figure 4.

      6. Check the size of DNA peaks on the Experion gel image. Make sure there is no visible peak around 100 bp (primer dimer). If it is present, purify the pooled libraries with AMPure XP beads again and repeat the quantification and Experion electrophoresis.

      7. Calculate the average DNA size (bp) of the library pool based on the Experion molarity (nM) and Experion concentration (ng/µl) using the following equation. Do NOT use the Qubit concentration to calculate the average DNA size.



  3. Notes:

    1. It is important to set up the cDNA reaction in a separate room from where the PCR amplification takes place to avoid potential contamination from PCR products.

    2. Viral RNA in this protocol that have very little quantity may not be measurable. qRT-PCR is usually recommended to confirm the copy numbers of viral RNA before library prep. We recommend at least 1,000 copies of input viral RNA for each library prep. However, this protocol may still work with less than 1,000 copies of input viral RNA, with compromised sampling depth.

    3. Primer mix is only needed when multiplexed regions are sequenced. It is possible to use one set of cDNA/forward primers at 10 µM concentration when sequencing only one region.

    4. Multiplexed regions normally should not overlap as this may inhibit the efficiency of priming and/or amplification.

    5. Include one negative control and possibly one positive control for each batch of library prep.

    6. It is recommended to aliquot the RNAclean XP beads and the AMPure XP beads into 1 ml aliquots. It is very important to warm the beads at room temperature for 30 min before use.

    7. The ratio of the volume of beads to cDNA/DNA is 0.7. It is optimized to remove long Primer ID cDNA primers or PCR primers.

    8. We recommend a second round of cDNA purification for some clinical specimens, especially plasma samples, to remove inhibitors of the downstream amplification. After the cDNA is eluted in water (in this case, use 40 µl water), add 28 µl of new RNAclean XP beads and purify the cDNA for a second time.

    9. Residual beads in the cDNA or PCR products eluent won’t affect the downstream amplification.

    10. First and second round PCR amplifications use two different KAPA PCR kits. The enzyme and buffer tubes look similar. It is very important to use the right buffer with the right enzyme for PCR 1 and PCR 2.

    11. Save the first round PCR product in the 1.5 ml tube in -20 °C, in case it is needed to repeat PCR2.

    12. Make sure to excise the fragment of the right size during gel extraction. Occasionally larger fragments (1.5-3 kb) can be present on a gel, which should not be extracted.

    13. Library concentration normally should be at least 20 ng/µl. Discard libraries with concentration less than 10 ng/µl as they are less likely to have high quality sequences.

    14. Qubit 2.0 fluorometer has been discontinued. Qubit 4.0 fluorometer is available at the time this protocol is written.

    15. Qubit dsDNA HS Assay kit is not recommended to quantify the libraries. The HS kits have an upper detection limit of 100 ng/µl. In some cases the library concentration can be higher than 100 ng/µl.

    16. The Bio-Rad Experion system is used in this protocol but it is close to obsolete. A better substitution is Agilent TapeStation System (Agilent, catalog number: G2991AA). The TapeStation system can directly measure the average DNA size of the library pool.



      Figure 4. An example of Experion gel image


  4. MiSeq sequencing run

    The MiSeq is an integrated instrument that performs clonal amplification, DNA sequencing based on sequencing-by-synthesis, and data analysis with base calling, alignment, variant calling, and reporting in a single run. In our protocol, we use the MiSeq reagent kit V3 (600 cycle) for sequencing. Figure 5 shows the MiSeq reagent cartridge and content by well numbers. This step is modified based on the MiSeq V3 protocol.



    Figure 5. MiSeq Reagent Cartridge and content by well numbers


    1. Prepare the reagent cartridge.

      1. Remove the reagent cartridge from -20 °C storage.

      2. Place the reagent cartridge in a water bath containing only enough room temperature, deionized water to submerge the base of the reagent cartridge. Do not allow the water to exceed the maximum water line printed on the reagent cartridge.

      3. Allow the reagent cartridge to thaw in the room temperature water bath for approximately 60 min or until completely thawed.

      4. Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge, if necessary.

      5. Invert the reagent cartridge ten times to mix the thawed reagents, and visually inspect that all positions are thawed.

      6. Visually inspect the reagent marked IMT (incorporation mix) to make sure that it is fully mixed and free of precipitates.

      7. Gently tap the cartridge on the bench to dislodge water from the base of the cartridge and reduce air bubbles in the reagents.

        Note: The MiSeq sipper tubes go to the bottom of each plate well to aspirate the reagents so it is important that there are no air bubbles stuck at the bottom of the reagent wells.

      8. Place the reagent cartridge on ice or set aside at 2 °C to 8 °C until you are ready to load the sample onto the reagent cartridge and set up the run.

    2. Prepare the libraries.

      1. Denature DNA

        1. Prepare a fresh dilution of 0.2 N NaOH on the day of sample loading. This is essential in order to completely denature samples for cluster generation on the MiSeq. Always start with a concentrated stock of NaOH that is 10 N or higher, or you may get a decrease in cluster density.

        2. Calculate the molarity of the pooled library based on the Qubit concentration and average DNA size, dilute the pooled library to 4 nM.

        3. Combine the following volumes of sample DNA and 0.2 N NaOH in a microcentrifuge tube:

          1. 4 nM sample DNA (5 µl)

          1. 0.2 N NaOH (5 µl)

        4. Vortex briefly to mix the sample solution, and then briefly centrifuge the sample solution.

        5. Incubate for five minutes at room temperature to denature the DNA into single strands.

        6. Add the following volume of pre-chilled HT1 to the tube containing denatured DNA to result in a 20 pM denatured library:

          1. Denatured DNA (10 μl)

          2. Pre-chilled HT1 (990 μl) (Shipped frozen in the MiSeq reagent cartridge box)

        7. Place the denatured DNA on ice until you are ready to proceed to final dilution.

      2. Dilute Denatured DNA

        1. Dilute the denatured DNA to 8 pM concentration using the following volumes:

          1. 20 pM DNA (400 µl)

          2. Pre-chilled HT1 (600 µl)

        2. Vortex briefly to mix the DNA solution.

        3. Pulse centrifuge the DNA solution.

        4. Place the denatured and diluted DNA on ice until you are ready to load your samples onto the MiSeq reagent cartridge directly or after adding the PhiX control.

      3. Denature and Dilute PhiX control

        1. PhiX is used as an internal sequencing control provided by Illumina. Use the following instructions to denature and dilute the 10 nM PhiX library to 8 pM. This should result in a cluster density of 750-850 K/mm2.

        2. Combine the following volumes to dilute the PhiX library to 2 nM:

          1. 10 nM PhiX library (2 μl)

          2. 10 mM Tris-HCl, pH 8.5 with 0.1% Tween 20 (8 μl)

        3. Combine the following volumes of 2 nM PhiX library and 0.2 N NaOH in a microcentrifuge tube to result in a 1 nM PhiX library:

          1. 2 nM PhiX library (10 μl)

          2. 0.2 N NaOH (10 μl)

        4. Vortex briefly to mix the 1 nM PhiX library solution.

        5. Briefly centrifuge the template solution.

        6. Incubate for five minutes at room temperature to denature the PhiX library into single strands.

        7. Add the following volume of pre-chilled HT1 to the tube containing denatured PhiX library to result in a 20 pM PhiX library.

          1. Denatured PhiX library (20 μl)

          2. Pre-chilled HT1 (980 μl)

        8. Dilute the denatured 20 pM PhiX library to 8 pM as follows:

          1. 20 pM denatured PhiX library (400 μl)

          2. Pre-chilled HT1 (600 μl)

      4. Mix Sample Library and PhiX Control

        1. Combine the following volumes of denatured PhiX control library and your denatured sample library to result in a 15% volume ratio:

          1. 8 pM PhiX control library (105 μl)

          2. Denatured sample library (595 μl)

        2. Set the combined sample library and PhiX control aside on ice until you are ready to load it onto the MiSeq reagent cartridge.

    3. Load Sample Libraries onto Cartridge

      1. Use a separate, clean, and empty 1 ml pipette tip to pierce the foil seal over the reservoir labeled Load Samples, position 17 on the reagent cartridge.

      2. Pipette 700 μl of your sample libraries into the Load Samples reservoir.

    4. Load Old Nextera Primer onto Cartridge.

      1. Old Nextera Primer is a custom Read 1 primer that gets spiked into position 12 on the reagent cartridge. Old Nextera Primer sequence can be found in Table 1.

      2. In a new microcentrifuge tube, add 3.8 µl of 100 µM Old Nextera Primer, and place on ice.

      3. Use a separate, clean, and empty 1 ml pipette tip to pierce the foil seal over the reservoir position 12 on the reagent cartridge.

      4. Pipette approximately 500 μl of the Read 1 Primer Mix out of the cartridge using a 1 ml serological pipette.

      5. Add the Read 1 Primer Mix to the tube with the Old Nextera Primer.

      6. Vortex briefly to mix the primer solution, and then briefly centrifuge the solution.

      7. Pipette the entire primer solution back into position 12 on the reagent cartridge using a 1 ml pipette tip.

        Note: Do not pierce any other reagent positions, they will be pierced when the cartridge is loaded on the instrument.

      8. Proceed directly to the run setup steps using the MiSeq Control Software (MCS) interface. Index sequences required for MCS can be found in Table 2.

    Notes:

    1. In the Illumina Experiment Manager (the software where you create the sample sheet) there is an option to select “Custom Primer for Read 1”. Since we are spiking in the custom primer into position #12, we do NOT select that option.

    2. Double check the primer sequence is correct and it is diluted to 100 µM. It is important that the custom primer gets mixed well with the other primers in position #12. We recommend pipetting 3.8 µl of primer into a new Eppendorf tube. Then remove the contents of position #12 and add them to the Eppendorf tube. Vortex to mix well. Then pipette the entire contents back into position #12 of the reagent cartridge. Tap the cartridge to make sure the liquid falls to the bottom of the tube.

    3. Do NOT add Old Nextera Primer into Positions 18 to 20 (custom sequencing primers).

    4. It is very important that the stock solution of NaOH is 10 N or higher. Some people try to use either 5 N or 2 N stock. For some reason these lower concentration stocks don’t work as well for denaturation and the 0.2 N dilution has to be made on the day of loading samples on the MiSeq.

    5. The denatured 20 pM PhiX library can be stored up to three weeks if stored at -15 °C to -25 °C. After three weeks, cluster numbers tend to decrease.

Data analysis

  1. Use the Illumina bcl2fastq pipeline (v.2.20.0) for initial data processing. This pipeline will de-multiplex sequences based on their barcodes. In this experiment design, each barcoded library is a library constructed from an RNA specimen, either from the treatment group or from the control group. In this step, only the library barcodes are de-multiplexed. The region and Primer ID de-multiplexing will be done in the following steps.

  2. Use TCS pipeline (v.1.3.8) to de-multiplexed each target region and create template consensus sequences. There are two ways to process it.

    1. (Recommended) Use TCS web app (https://tcs-dr-dept-tcs.cloudapps.unc.edu/) to process the raw MiSeq sequences automatically. The web app asks for the information of each sequenced regions including a name, the cDNA primer sequence and the first round PCR forward primer sequence. Choose direct upload, UNC Longleaf sequence repo or Dropbox to upload the raw MiSeq sequences. The processed sequences will be returned to the email address provided by the user. The process time is from 20 min to 2 h, based on the size of files for each batch submitted.

    2. Use the TCS pipeline script (https://github.com/SwanstromLab/PID/blob/master/TCS.rb) to process the MiSeq raw sequence data manually, following the instructions in the script. It is recommended to use the file-sorting and logging script in the TCS pipeline ‘log_mutli.rb’ (https://github.com/SwanstromLab/PID/blob/master/log_multi.rb) to sort the files after running the TCS pipeline. The script can be run on the Linux/MacOS platform using Terminal and Ruby without additional support, and in Windows platform with terminal and Ruby installed.

  3. Use the ‘end_join.rb’ script (https://github.com/SwanstromLab/PID/blob/master/end_join.rb) in the TCS pipeline to join the paired-end sequences.

  4. The number of TCS is an important parameter that reveals the sampling depth of sequencing as the number of initial templates actually queried.

  5. Use the RUBY package ‘viral_seq’ (1.0.8) to calculate the mutation rates of each specimen. An executable script can be found in the TCS pipeline (https://github.com/SwanstromLab/PID/blob/master/mut_table.rb). This script compares each individual sequence with a sample consensus sequence and generates a table listing frequencies of each type of substitutions.


Notes:

  1. The newer version of the TCS pipeline (version ≥ 2.0.0) is included in the RUBY package ‘viral_seq’ (version ≥ 1.0.8) as an executable command line tool. It has several major updates including the end-joining functions along with the TCS creation, thus it is recommended for future use.

  2. The authors dedicated to improve the bioinformatics analysis pipelines. Users are welcome to contact the authors for any related questions.

Acknowledgments

We would like to acknowledge the following funding sources: National Institutes of Allergy and Infectious Disease R01 AI132178 awarded to T.P.S. and R.S.B,. and NIAID 5R01 AI140970 awarded to R.S.. We thank the UNC High Throughput Sequencing Facility for help developing this protocol. This protocol was used in our previously published work (Sheahan et al., 2020).

Competing interests

The University of North Carolina is pursuing IP protection for Primer ID and R.S. has received nominal royalties.

References

  1. Abrahams, M. R., Joseph, S. B., Garrett, N., Tyers, L., Moeser, M., Archin, N., Council, O. D., Matten, D., Zhou, S., Doolabh, D., Anthony, C., Goonetilleke, N., Karim, S. A., Margolis, D. M., Pond, S. K., Williamson, C., and Swanstrom, R. (2019). The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation. Sci Transl Med 11(513): eaaw5589.
  2. Adewumi, O. M., Dukhovlinova, E., Shehu, N. Y., Zhou, S., Council, O. D., Akanbi, M. O., Taiwo, B., Ogunniyi, A., Robertson, K., Kanyama, C., Hosseinipour, M. C. and Swanstrom, R. (2020). HIV-1 Central Nervous System Compartmentalization and Cytokine Interplay in Non-Subtype B HIV-1 Infections in Nigeria and Malawi. AIDS Res Hum Retroviruses 36(6): 490-500.
  3. Clutter, D. S., Zhou, S., Varghese, V., Rhee, S. Y., Pinsky, B. A., Jeffrey Fessel, W., Klein, D. B., Spielvogel, E., Holmes, S. P., Hurley, L. B., Silverberg, M. J., Swanstrom, R. and Shafer, R. W. (2017). Prevalence of Drug-Resistant Minority Variants in Untreated HIV-1-Infected Individuals With and Those Without Transmitted Drug Resistance Detected by Sanger Sequencing. J Infect Dis 216(3): 387-391.
  4. Council, O. D., Zhou, S., McCann, C. D., Hoffman, I., Tegha, G., Kamwendo, D., Matoga, M., Kosakovsky Pond, S. L., Cohen, M. S. and Swanstrom, R. (2020). Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without Sexually Transmitted Infection-Associated Urethritis. J Virol 94(12): e00151-20.
  5. Dennis, A. M., Zhou, S., Sellers, C. J., Learner, E., Potempa, M., Cohen, M. S., Miller, W. C., Eron, J. J., and Swanstrom, R. (2018). Using Primer-ID Deep Sequencing to Detect Recent Human Immunodeficiency Virus Type 1 Infection.J Infect Dis 218(11): 1777-82.
  6. Jabara, C. B., Jones, C. D., Roach, J., Anderson, J. A. and Swanstrom, R. (2011). Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc Natl Acad Sci U S A 108(50): 20166-20171.
  7. Keys, J. R., Zhou, S., Anderson, J. A., Eron, J. J., Jr., Rackoff, L. A., Jabara, C. and Swanstrom, R. (2015). Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain. AIDS Res Hum Retroviruses 31(6): 658-668.
  8. Lee, S. K., Zhou, S., Baldoni, P. L., Spielvogel, E., Archin, N. M., Hudgens, M. G., Margolis, D. M. and Swanstrom, R. (2017). Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay. J Acquir Immune Defic Syndr 74(2): 221-228.
  9. Lewis, C. A., Jr., Crayle, J., Zhou, S., Swanstrom, R. and Wolfenden, R. (2016). Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history. Proc Natl Acad Sci U S A 113(29): 8194-8199.
  10. Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., Lin, D., Lu, L. and Law, M. (2012). Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012: 251364.
  11. Sheahan, T. P., Sims, A. C., Zhou, S., Graham, R. L., Pruijssers, A. J., Agostini, M. L., Leist, S. R., Schafer, A., Dinnon, K. H., 3rd, Stevens, L. J., Chappell, J. D., Lu, X., Hughes, T. M., George, A. S., Hill, C. S., Montgomery, S. A., Brown, A. J., Bluemling, G. R., Natchus, M. G., Saindane, M., Kolykhalov, A. A., Painter, G., Harcourt, J., Tamin, A., Thornburg, N. J., Swanstrom, R., Denison, M. R., and Baric, R. S. (2020). An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med 12(541): eabb5883.
  12. Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. and Madden, T. L. (2012). Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13: 134.
  13. Zhou, S., Bednar, M. M., Sturdevant, C. B., Hauser, B. M. and Swanstrom, R. (2016). Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions.J Virol 90(16): 7142-7158.
  14. Zhou, S., Jones, C., Mieczkowski, P. and Swanstrom, R. (2015). Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations. J Virol 89(16): 8540-8555.
  15. Zhou, S., Williford, S. E., McGivern, D. R., Burch, C. L., Hu, F., Benzine, T., Ingravallo, P., Asante-Appiah, E., Howe, A. Y. M., Swanstrom, R. and Lemon, S. M. (2018). Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus. Antiviral Res 158: 45-51.

简介

[摘要]下一代测序(NGS)已成为生物医学研究的重要工具。结合MiSeq平台的Primer ID方法克服了PCR错误的局限性,并揭示了群体测序的真实采样深度,使其成为研究潜在的广谱抗病毒剂对RNA病毒的诱变作用的理想工具。在本报告中,我们描述了一种使用引物ID测序的方案,用于研究体外细胞培养模型和体内小鼠模型中冠状病毒基因组中抗病毒药诱导的突变。在最初的反转录步骤中,病毒RNA或总肺组织RNA用含Primer ID的cDNA引物标记,然后进行两轮PCR扩增病毒序列并整合测序适配器。使用MiSeq平台对纯化和合并的文库进行测序。测序数据使用模板共有序列(TCS)网络应用处理。引物ID方法提供了一种精确的测序方案,可以测量病毒RNA基因组和宿主mRNA中的突变错误率。测序结果表明,β-D-N4-羟基胞嘧啶核苷(NHC)大大提高了病毒RNA基因组中的过渡取代率,但并未显着提高颠覆取代率,并且发现胞嘧啶(C)至尿苷(U)是最常见的突变。


[背景]下一代测序(NGS)已被广泛应用在生物医学研究中使用在过去十年。当应用NGS研究宿主内病毒种群的RNA病毒时,需要考虑对文库制备和测序方案的修改。样本之间的病毒滴度(或病毒载量)差异很大。传统的NGS平台在测序运行中需要1-500 ng的DNA(或RNA),但在大多数情况下,临床样品中的病毒RNA少于100 fg ,需要先使用逆转录酶将病毒RNA转化为cDNA ,然后进行一轮或两轮PCR扩增,以产生足够的材料进行测序。然而,PCR扩增的广泛循环将引起核苷酸错误掺入,重组和扩增偏倚。此外,NGS平台具有相对较高的错误率,给所得序列增加了额外的不确定性(Liu等人,2012)。

我们开发了Primer ID NGS方法来克服传统NGS方法的错误和偏见(Jabara等,2011; Zhou等,2015)。在最初的cDNA合成步骤中,我们使用带有11个碱基的简并核苷酸嵌入片段(引物ID)的cDNA引物为每个病毒RNA模板添加唯一ID,这是添加唯一分子标识符(UMI)的一种方法)。引物ID在整个下游扩增和测序过程中都会进行。测序后,将所有具有相同ID的原始序列折叠起来,制成模板共有序列(TCS)。每个TCS都链接到在初始cDNA合成步骤中查询的原始模板,并且TCS的数量显示了病毒种群的测序采样深度。通过从每个病毒模板的多个原始测序读数中构建共有序列,我们可以大大减少PCR和测序错误。我们已经展示了Primer ID方法可以将测序错误极大地减少到10,000个核苷酸中的1个(与原始测序读数相比减少了100倍),并且还揭示了病毒种群的真实​​采样深度,从而可以精确地测量取代岭TES和多样性群体中的病毒基因组(图1) (周等人,2015) 。我们进一步开发了一种多重引物ID方法,允许在单个cDNA合成/ PCR中对病毒基因组的多个区域进行测序(图2)(Dennis et al。,2018)。


图1.入门手册ID方法。一。引物ID引物的结构及其与病毒RNA模板的结合。乙。从原始序列读取中创建模板共有序列的示例。





图2.多重引物ID测序的文库制备步骤。生物序列区为蓝色,引物ID区为黄色,正向间隔区为橙色,MiSeq条码序列区为紫色。



由于该方法能够准确表征单个病毒基因组,因此是研究宿主内RNA病毒种群的理想方法。我们已经使用这种方法来确定HIV宿主内病毒种群的遗传结构(Zhou等人,2016; Lee等人,2017; Dennis等人,2018; Abrahams等人,2019; Adewumi等人) 。2020年,理事会等,2020) ,在HIV和HCV研究耐药突变(杂波等人,2017年;周某等人,2018;钥匙。等人,2015年),并检测核苷酸突变率加热后的单链DNA分子中的蛋白质(Lewis等人,2016)。

在本报告中,我们描述了使用引物ID MiSeq测序方法检测EIDD-1931(β-D-N4-羟基胞苷,NHC)和EIDD-2 80 1(EIDD-1931的口服生物利用度前体药物和根据体外细胞培养和体内小鼠模型,在中东呼吸综合征冠状病毒(MERS- CoV )基因组中也被识别为MK-4482或Molnupiravir )。在细胞培养测序实验中,我们使用了多重引物ID协议在单个文库制备和测序反应中对MERS- CoV ORF 1b中的多个区域进行测序,而在对MERS- CoV感染小鼠的总肺RNA进行测序时,我们在单个文库制备和测序反应中使用针对MERS- CoV基因组和两个选定的小鼠mRNA的多重Primer ID方案,其中我们可以直接比较EIDD-2801诱导的对MERS- CoV基因组和宿主mRNA的诱变作用。该方案不仅限于MERS- CoV和EIDD-1931研究,而且通常可用于检测病毒RNA突变率。

关键字:冠状病毒, 抗病毒药, 新一代测序, 引物 ID, 突变


材料和试剂
1. P ipette提示     

2.收集管(QIAGEN,目录号:19201,在室温下保存)     

3. Qubit分析管(ThermoFisher Scientific,Invitrogen,目录号:Q32856,在室温下保存)     

4. SuperScript III一步式RT-PCR系统(ThermoFisher Scientific,Invitrogen,目录号:12574026,存储在-20 °C )     

5.在-20 °C下保存的Tris-HCl缓冲液中100 µM的引物ID引物(Integrated DNA Technologies)     

6. SuperScript III逆转录酶(ThermoFisher Scientific,Invitrogen,目录号:18080085,存储在-20 °C )     

7. RNaseOUT重组核糖核酸酶抑制剂(ThermoFisher Scientific,Invitrogen,目录号:10777019,在-20 °C储存)     

8.核糖核酸酶H (ThermoFisher Scientific,Invitrogen,目录号:18021071,在-20 °C下储存)     

9. KAPA2G耐用的PCR试剂盒(带有dNTP的Hotstart PCR试剂盒)(Roche,目录号:KK5516,存储在-20 °C )     

10. KAPA2G HiFi HotStart (带有dNTP )(Roche,目录号:KK2502,存储在-20 °C ) 

11. RNAClean X P(Beckman Coulter,目录号:A63987,在4 °C下储存) 

12. AMPure XP(贝克曼库尔特,目录号:A63881,在4 °C下储存) 

13.乙醇200证明(Decon实验室,目录号:2716) 

14.不含DNase / RNase的水 

15. MinElute凝胶提取试剂盒(QIAGEN,目录号:28606,用于MinElute色谱柱,在4 °C下储存,在室温下用于其他试剂) 

16. Qubit dsDNA BR检测试剂盒(ThermoFisher Scientific,Invitrogen,目录号:Q32853,标准品1和标准品2在4 °C下储存,其他试剂在室温下保存) 

17. Experion DNA 12K分析试剂盒(Bio- R ad,目录号:7007108,芯片在室温下保存,其他试剂在4 °C下保存) 

18. MiSeq试剂盒v3(600个循环)(Illumina ,目录号:MS-102-3003,对于第1盒,在-20 °C下存储;对于第2盒,在4 °C下存储) 

19.氢氧化钠(西格玛奥德里奇,目录号:221465,在室温下保存) 

20.盐酸Tris (Sigma-Aldrich,目录号:10812846001,在室温下保存) 

21. PhiX Control V3(Illumina ,目录号:FC-110-3001,存储在-20 °C ) 

22. 10 mM Tris-HCl,含0.1%TWEEN 20(Teknova ,目录号:T7724,在室温下保存) 



设备


皮pettes
磁力架
热循环仪(ThermoFisher Scientific ,Applied Biosystems ,目录号:4384638 )
离心机(Eppendorf ,型号:5424)
DynaMag-2磁铁(ThermoFisher Scientific,Invitrogen,目录号:12321D)
Qubit 2.0荧光计(ThermoFisher Scientific,Invitrogen,目录号:Q32866)
Experion电泳站(Bio- R ad,目录号:7007010)
MiSeq系统(Illumina ,目录号:SY-410-1003)


软件


Primer-BLAST(NCBI,https://www.ncbi.nlm.nih.gov/tools/primer-blast/)(Ye et al。,2012)
bcl2fastq管道(v.2.20.0)(Illumina ,https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html)
TCS Web应用程序(UNC,https://tcs-dr-dept-tcs.cloudapps.unc.edu/)
TCS管道(v.1.3.8)(UNC,https://github.com/SwanstromLab/PID)
RUBY软件包viral_seq (v.1.0.8)(UNC,https://github.com/ViralSeq/viral_seq)


程序


我们首先设计用于多重引物ID文库制备的引物。从组织培养上清液中提取病毒RNA或从肺组织中提取总RNA后,我们使用来自每个标本的多重引物ID库制备方案构建MiSeq测序库。多重引物ID协议使我们可以在一个反应中构建病毒RNA多个区域的测序文库。每次测序我们最多收集24个条形码文库(来自24个不同的标本)。我们使用Illumina MiSeq V3试剂盒(600个循环)进行测序。我们选择配对末端测序,以具有更长的读取长度,并且比单末端测序具有更好的重叠区域纠错能力。


引物ID cDNA引物和PCR引物设计
使用NCBI Primer-BLAST设计引物序列。
使用参考基因组MERS- CoV NC_019843的ORF1b区作为Primer-BLAST界面中的“ PCR模板”来设计MERS- CoV的引物。使用具有四三肽重复序列2(Ifit2,参考号:NM_008332)的小鼠干扰素诱导的蛋白质,具有四三肽重复序列3(Ifit3,参考号:NM_010501),ISG15泛素样修饰剂(Isg15,参考号:NM_015783),干扰素诱导的蛋白具有四三肽重复序列1(Ifit1,参考号:NM_008331)和趋化因子(CXC基序)配体10(Cxcl10,参考号:NM_021274)基因组的蛋白质作为模板来设计小鼠mRNA的引物。
将PCR产物大小设置为最小值:400 bp ,最大值:600 bp。
将底漆熔化温度设置为最低:52,选择:55,最高:58。
为每个模板区域选择3-4对引物。
使用SuperScript III单步RT-PCR系统以每个区域的最佳扩增效率测试引物。
使用上述组织培养物中的MERS- CoV病毒RNA进行MERS- CoV引物测试。使用提取的小鼠总肺RNA作为小鼠mRNA引物测试的模板。在无DNase / RNase的水中,以1:10的滴度对模板RNA进行连续滴定,作为RT-PCR模板。稀释至10 -6 。
使用以下配方在PCR试管中制备一个单步RT-PCR混合物(总体积为10 µl )。
5 µl 2 ×缓冲液


1.8 µl不含DNase / RNase的水


0.4 µl正向引物(20 µM)


0.4 µl反向R(20 µM)


0.4 µl酶混合物


2 µl模板RNA


将以下热循环仪条件用于RT-PCR。
50 °C 30分钟
94 °C 2分钟
40个周期的:
94 °C持续15 s


55 °C持续30 s


68 °C持续30 s


68 °C 5分钟
使用1%琼脂糖凝胶进行凝胶电泳,检查PCR产物。
图3显示了来自测试的MERS- CoV引物的扩增子的示例凝胶图像。
 



图3.用于引物设计的示例性一步RT-PCR凝胶图像。测试了针对相同的MERS- CoV基因组的三组引物。将MERS- CoV RNA的连续稀释液稀释至10 -6稀释液来测试引物。使用SSIII一步PCR系统进行扩增。通过用1%琼脂糖凝胶的凝胶电泳检查PCR产物。引物组2具有最佳扩增效率,因此被选作下游引物ID引物设计。NC,无反应控制。


设计MiSeq文库制备用引物ID cDNA引物和其他引物。
通过将引物ID(11个碱基长的序列的简并片段)和衔接子序列连接到反向引物上,设计cDNA引物。
前进1的引物第一轮PCR是由在3'与在步骤所设计的正向引物测序端)在5'接合的共同序列“GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNN”设计。连接的引物序列中的Ns是随机序列,以提高测序质量。
的1的反向引物ST轮PCR被设计抵抗引物ID的衔接子序列部分的cDNA的引物,并且是不管通用区域(表1)。
2个第二轮PCR引物被设计成包括MiSeq与条形码测序衔接。
为了引物上设计上述小号TEP小号甲1A- A1 d从集成DNA技术(IDT)。要求手工混合前体以降低cDNA引物序列的变性区,以使碱基在每个位置上均匀分布。
表1显示了此方案中使用的引物的完整列表,包括用于Primer ID MiSeq文库制备的引物和定制的MiSeq测序引物。表2显示了索引引物的序列。
笔记:


引物-BLAST不是设计引物所必需的。引物可以基于已知的参考基因组进行手动设计。
在引物ID cDNA引物合成之前,先手动混合核苷酸前体对于确保简并序列在简并区块每个位置上碱基(A,C,G,T )的分布均很重要。


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说明:logonew                                                             



表1.用于MiSeq文库制备和测序的引物。具有基因特异性区域的引物序列以蓝色突出显示。在设计其他病毒的引物时,可以将它们替换为其他基因特异性引物序列。


底漆


5'-3'序列


评论


41R_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGT ATGACCTTCCTGTTGCTTCT


cDNA引物。在参考基因组上靶向20331-20350。


nsp10_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGT CCTAAAGACGACATCAGTGG


cDNA引物。在参考基因组上靶向13488-13507。


nsp12_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGT ATAGCCAAAGACACAAACCG


cDNA引物。在参考基因组上靶向15983-16002。


nsp14_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGT GAACATCGACAAAGAAAGGG


cDNA引物。在参考基因组上靶向18715-18734。


ifit3_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGT TTCAGCCACTCCTTTATCCC


cDNA引物。靶向小鼠IFIT3 mRNA。


isg15_PID11 *


GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGT GGGGCTTTAGGCCATACTC


cDNA引物。靶向小鼠ISG15 mRNA。


41F_AD


GCCTCCCTCGCGCGCCATCAGAGATGTGTATAAGAGACAGNNNN GCTACAAGTTCGTCCTTTGG


1第一轮PCR的正向引物。在参考基因组上靶向19812-19831


nsp10_AD


GCCTCCCTCGCGCGCCATCAGAGATGTGTATAAGAGACAGNNNN TGCTCAGGTGCTAAGCGAAT


1第一轮PCR的正向引物。在参考基因组上靶向12983-13002


nsp12_AD


GCCTCCCTCGCGCGCCACAGAGATGTGTATAAGAGACAGNNNN ATAGGCTTCGATGTTGAGGG


1第一轮PCR的正向引物。在参考基因组上靶向15388-15407


nsp14_AD


GCCTCCCTCGCGCGCCATCAGAGATGTGTATAAGAGACAGNNNN ATTGCAAGCTGGTTCTAACA


1第一轮PCR的正向引物。在参考基因组上靶向18260-18279


ifit3_AD


GCCTCCCTCGCGCGCCACAGAGATGTGTATAAGAGACAGNNNN CGATCCACAGTGAACAACAG


1第一轮PCR的正向引物。靶向小鼠IFIT3 mRNA。


isg15_AD


GCCTCCCTCGCGCGCCACAGAGATGTGTATAAGAGACAGNNNN TGGGACCTAAAGGTGAAGATG


1第一轮PCR的正向引物。靶向小鼠ISG15 mRNA。


表1.续


转接器R


GTGACTGGAGTTCAGACGTGTGCTC


1第一轮PCR反向引物


通用适配器


AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCGCCATCAGAGATGTG


2第二轮PCR使用正向引物的Illumina衔接子序列


索引适配器**


CAAGCAGAAGACGGCATACGAGAT NNNNNN GTGACTGGAGTTCAGACGTGTGCTC


2第二轮PCR与反向引物的Illumina衔接子序列和指标(NNNNNNN)


老Nextera


GCCTCCCTCGCGCGCCACAGAGATGTGTATAAGAGACAG


定制测序引物


* cDNA引物具有一个简并核苷酸(Ns)区块作为引物ID。


** Indexed Adapter引物中的Ns是一组24个预先设计的条形码,用于多路复用MiSeq运行的样品。请参阅表2中的索引序列和适配器序列。


 



表2.索引的适配器序列。请注意,索引序列是适配器序列中突出显示的(红色)区域的反向互补序列


索引适配器


指数


索引序列


顺序(5'-3')


PCR引物,索引1


1个


ATCACGA


CAAGCAGAAGACGGCATACGAGA T CGTGAT GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引2


2个


GA


CAAGCAGAAGACGGCATACGAGA T ACATCG GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引3


3


塔加卡


CAAGCAGAAGACGGCATACGAGA T GCCTAA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引4


4


泰格卡


CAAGCAGAAGACGGCATACGAGA TT GGTCA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引5


5


ACAGTGA


CAAGCAGAAGACGGCATACGAGA T CACTGT GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引6


6


卡卡塔


CAAGCAGAAGACGGCATACGAGA T ATTGGC GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引7


7


卡加塔卡


CAAGCAGAAGACGGCATACGAGA T GATCTG GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引8


8


ACTTGAA


CAAGCAGAAGACGGCATACGAGA T TCAAGT GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引9


9


GATCAGA


CAAGCAGAAGACGGCATACGAGA T CTGATC GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引10


10


塔格塔


CAAGCAGAAGACGGCATACGAGA T AAGCTA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引11


11


GGCTACA


CAAGCAGAAGACGGCATACGAGA T GTAGCC GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引12


12


CTTGTAA


CAAGCAGAAGACGGCATACGAGA T TACAAG GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引13


13


TCCATAA


CAAGCAGAAGACGGCATACGAGA T TATGGA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引14


14


GTACTAA


CAAGCAGAAGACGGCATACGAGA T TAGTAC GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引15


15


阿格塔阿


CAAGCAGAAGACGGCATACGAGA T TACTGT GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引16


16


卡塔加


CAAGCAGAAGACGGCATACGAGA T CATGAG GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引17


17


阿加塔加


CAAGCAGAAGACGGCATACGAGA T TATCGT GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引18


18岁


TGCAGAA


CAAGCAGAAGACGGCATACGAGA T TCTGCA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引19


19


TTCATAA


CAAGCAGAAGACGGCATACGAGA T TATGAA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引20


20


TGCTGTA


CAAGCAGAAGACGGCATACGAGA T ACAGCA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引21


21岁


塔塔卡


CAAGCAGAAGACGGCATACGAGA T GTGATA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物索引22


22


TGGATAA


CAAGCAGAAGACGGCATACGAGA T TATCCA GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引23


23


CGCATTA


CAAGCAGAAGACGGCATACGAGA T AATGCG GTGACTGGAGTTCAGACGTGTGCTC


PCR引物,索引24


24


贸发会议


CAAGCAGAAGACGGCATACGAGA T TAAGGC GTGACTGGAGTTCAGACGTGTGCTC


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说明:logonew               

引物ID MiSeq库制备
cDNA合成
使用从感染细胞的细胞培养上清液中提取的病毒RNA或感染小鼠的肺组织总RNA作为扩增模板。cDNA合成步骤应在BSL2 +设施中进行,直到核糖核酸酶H步骤之后。
完全解冻,涡旋并旋转5 ×缓冲液,dNTP和DTT。
使基因从100μM引股票底漆工作组合。引物混合物中每种引物的终浓度为10 µM。
确定测序区域。
将10 µl的每种cDNA引物(最多5个不同的引物进行多重化)添加到Eppendorf微量离心管中,并添加无DNase / RNase的水至总体积为100 µl。
如果需要更大体积的引物混合物,请按比例增加引物和无DNase / RNase的水的量。
吸取以下成分到0.5 ml无RNase的PCR管中。
2 µl dNTP混合物(每个10 mM )


1 µl引物ID cDNA引物混合物(每个10 µM)


23 µl RNA模板(vRNA或总肺RNA)


将PCR试管在热循环仪上于65°C加热5分钟,然后在盖关闭的情况下冷却至4 °C 2分钟(盖温度为105 °C )。
将以下组件添加到管中。如果分批进行cDNA合成,则要进行预混,然后将14 µl的混合物加到每个试管中。
8 µl 5 ×缓冲液


2 µl DTT(100 mM )


2微升RNaseOUT (40 U /微升)


2 µl SSIII逆转录酶(200 U / µl)


通过上下移液几次将其混合均匀。将试管转移到热循环仪上。使用以下热循环仪程序进行cDNA合成。
55 °C 1小时


50 °C 1小时


70 °C 15分钟


到每个管中,加入0.5微升(1U)Ribonucleas Ë H,孵育在37 ℃进行20分钟。此步骤后,可以在常规工作台(BSL1)上处理cDNA 。 
净化的cDNA与RNAClean XP。
通过涡旋重悬珠并取出等分试样。使用前,请在室温下至少保存30分钟。
通过涡旋彻底重悬be be广告。向1.5 ml管中加入28 µl RNAClean XP珠。将所有的cD NA反应(40.5 µl)转移到每个有珠子的管中。
              将RNAClean XP和移液器混合15次,充分混合样品。涡旋是不推荐使用。让试管在室温下孵育20分钟。如果样品接触到试管的上部,请短暂旋转。
将试管放在磁力试管架上5分钟,以将小珠从溶液中分离出来。
缓慢地从管中吸出已清除的溶液并丢弃。应在试管位于机架上时执行此步骤。请勿干扰已在管子侧面形成斑点的磁珠。
              分配400微升70%乙醇到管在室温下孵育30秒。吸出乙醇并丢弃。重复进行总共3次洗涤。重要的是要使用位于磁力架上的管子执行这些步骤。请勿打扰分离的磁珠。确保从井底除去所有乙醇,因为其中可能含有残留的污染物。
              打开盖子,让反应管在机架上风干10分钟。试管应风干,直到最后可见的乙醇痕迹蒸发。但是,样品过度干燥可能导致较低的回收率。
从磁力架上取下试管,然后上下吹打,将珠子重悬在24 µl不含DNase / RNase的水中。将试管放回机架上,静置3分钟。
当移液管位于磁性管架上时,从移液管中吸取洗脱液(23.5 µl)。
使用KAPA2G稳健PCR试剂盒进行第一轮PCR扩增。
在第一轮PCR中,我们使用针对基因组多个区域的正向引物混合物,扩增了从上一步获得的所有cDNA 。


用100 µM底漆储备液配制正向底漆。
根据cDNA引物放置的初始选择确定引物结合区域。
将每个正向引物(不超过5个引物)加10 µl到Eppendorf微量离心管中,并加入无DNase / RNase的水至100 µl的总体积。
如果需要更大体积的引物混合物,请按比例增加引物和无DNase / RNase的水的量。
使用前,将试剂(酶除外)完全融化,涡旋和旋转。
使用以下配方制作一个第一轮PCR混合物(总体积为50 µl)。
10 µl的5 ×缓冲液A


10 µl增强剂


1 µl dNTP


2.5 µl正向引物混合物


2.5 µl衔接子R引物


0.5 µl KAPA稳固聚合酶


23.5 µl模板cDNA


通过上下移液几次将其混合均匀。将试管转移到热循环仪上。将以下热循环仪程序用于第一轮PCR扩增。
95 °C 1分钟
25个周期:
95 °C持续15 s


58 °C持续60 s


72 °C持续30 s


72 °C 3分钟
使用AMPure XP清洁珠纯化PCR产品。
涡旋1毫升等分的AMPure XP清洁珠,并除去所需的体积。使用前,请在室温下至少保存30分钟。
将第一轮PCR反应转移到1.5 ml无DNase的试管中。
重悬珠子。在每个PCR反应中添加35 µl AMPure XP珠。
混合AMPure XP并涡旋彻底取样。让试管在室温下孵育5分钟,然后再进行下一步(在架子上孵育)。
              将试管放在磁力试管架上5分钟,以将小珠从溶液中分离出来。
              缓慢地从管中吸出已清除的溶液并丢弃。应在试管位于机架上时执行此步骤。请勿干扰已在管子侧面形成斑点的磁珠。
分配400微升的70%乙醇在室温下将试管孵育30秒。吸出乙醇并丢弃。重复两次,共洗涤一次。必须使用位于机架上的管子执行这些步骤。请勿打扰分离的磁珠。确保从井底除去所有乙醇,因为其中可能含有残留的污染物。
              打开盖子,让反应管在机架上风干10分钟。试管应风干,直到最后可见的乙醇痕迹蒸发。样品过度干燥可能导致较低的回收率。
              从架子上取下试管,然后上下吹打,将珠子重悬在50 µl不含DNase的水中。将试管放回机架上,静置3分钟。
当45 µl洗脱液位于磁性试管架上时,用移液管吸取。将洗脱液转移至新的1.5 ml微量离心管中。
使用KAPA2G HiFi PCR试剂盒进行第二轮PCR扩增。
使用前,请完成解冻,涡旋和旋转试剂(酶除外)的处理。
使用以下配方制作一轮第二轮PCR混合物(总体积为25 µl)。
10 µl的5 × KAPA HiFi缓冲液


1 µl dNTP


1 µl通用适配器


1 µl索引适配器


0.5 µl KAPA HiFi聚合酶


从第一轮PCR中提取2 µl纯化的DNA。


通过上下移液几次将其混合均匀。将试管转移到热循环仪上。使用以下热循环仪程序进行cDNA合成。
95 °C 2分钟
35个循环:
98 °C持续20 s


63 °C持续15 s


72 °C持续30 s


72 °C 3分钟
使用QIAGEN MinElute凝胶提取试剂盒对PCR产物进行凝胶纯化。
进行凝胶提取之前,请在1%琼脂糖凝胶上运行2 µl PCR产物,以检查文库的大小是否正确。
用溴化乙锭制成1.2%(w / v)琼脂糖凝胶(终浓度为0.2-0.5 µg / ml )。
将所有第二轮PCR产物加载到1.2%琼脂糖凝胶上。根据凝胶大小调整电压。理想的电场强度为4 V / cm。运行凝胶30分钟。
在非紫外线蓝光透照仪上精确切割目标DNA片段(500 bp至1 kb)。
              称量凝胶;将3体积的Buffer QG加到1体积的凝胶中。
              在50 °C下孵育10分钟以完全溶解凝胶片段。每2-3分钟涡旋一次以帮助溶解。
检查凝胶溶液的颜色(应为黄色,否则应加入10 µl 3 M pH 5.0的乙酸钠)。
              应用样品到的MinElute柱和离心机中以最大速度为1分钟。
将色谱柱转移到新的收集管中,弃去滤液,加入500 µl QG缓冲液并以最大速度离心1分钟。
将色谱柱转移到新的收集管中,弃去滤液,加入0.75 ml PE缓冲液,在室温下孵育5分钟,以最大速度离心1分钟。
将色谱柱转移到新的收集管中,弃去滤液,以最大速度离心3分钟。
将色谱柱放入新的1.5 ml试管中,加入10 µl EB缓冲液。静置4分钟,以最大速度离心2分钟。
量化,合并和QA / QC。
按照制造商的方案,使用Qubit dsDNA BR Assay试剂盒对每个凝胶纯化的文库进行定量。每次定量使用1 µl文库。
以等量合并量化的单个库。我们建议合并总共20-24个文库,每个文库具有100至150 ng DNA。如果计算出的容量超过实际容量,请使用库的最大可用容量。
按照上文“第一轮PCR”部分中的方案,使用AMPure XP珠子纯化文库。要使用的AMPure磁珠的体积等于合并库的体积乘以0.7。
按照制造商的规程,使用Qubit dsDNA BR Assay试剂盒定量纯化的合并文库。
按照制造商的规程,使用带有DNA 12k分析试剂盒的Experion自动电泳系统运行合并的文库。的凝胶图像的一个例子的Experion示于图URE 4。
检查Experion凝胶图像上DNA峰的大小。确保在100 bp附近(引物二聚体)没有可见的峰。如果存在,请再次用AMPure XP珠子纯化合并的文库,然后重复定量和Experion电泳。
使用以下公式,根据Experion摩尔浓度(nM )和Experion浓度(ng / µl )计算文库池的平均DNA大小(bp )。不要使用Qubit浓度来计算平均DNA大小。
 



笔记:


重要的是将cDNA反应设置在单独的房间中,在该房间中进行PCR扩增,以避免潜在的PCR产物污染。
此协议中的病毒RNA量很少,可能无法测量。通常建议使用qRT -PCR在文库制备之前确认病毒RNA的拷贝数。对于每种文库制备,我们建议至少输入1,000份输入病毒RNA。但是,此协议可能仍可用于少于1,000个输入病毒RNA副本,且采样深度受到影响。
仅当对多重区域进行测序时才需要引物混合。仅对一个区域测序时,可以使用一组10 µM浓度的cDNA /正向引物。
复用区域通常不应重叠,因为这可能会抑制启动和/或扩增的效率。
每一批文库制备应包括一个阴性对照,可能还包括一个阳性对照。
建议将RNAclean XP珠子和AMPure XP珠子等份分成1 ml等分试样。使用前,将珠子在室温下加热30分钟非常重要。
珠粒与cDNA / DNA的体积比为0.7。它经过优化,可去除长的Primer ID cDNA引物或PCR引物。
我们建议对某些临床标本,尤其是血浆标本,进行第二轮cDNA纯化,以去除下游扩增的抑制剂。将cDNA用水洗脱后(在这种情况下,使用40 µl水),添加28 µl新的RNAclean XP珠并再次纯化cDNA 。
cDNA或PCR产物洗脱液中的残留珠子不会影响下游扩增。
第一轮和第二轮PCR扩增使用两种不同的KAPA PCR试剂盒。酶和缓冲液管看起来相似。对于PCR 1和PCR 2,使用正确的缓冲液和正确的酶非常重要。
如果需要重复PCR2,请将第一轮PCR产物保存在-20 °C的1.5 ml管中。
确保在凝胶提取过程中切除适当大小的片段。有时,较大的片段(1.5-3 kb)可以存在于凝胶上,不应提取。 
库浓度通常应至少为20 ng / µl。丢弃浓度低于10 ng / µl的文库,因为它们不太可能具有高质量序列。
Qubit 2.0荧光计已停产。编写此协议时,可以使用Qubit 4.0荧光计。
不建议使用Qubit dsDNA HS Assay试剂盒定量文库。HS试剂盒的检测上限为100 ng / µl。在某些情况下,文库浓度可能高于100 ng / µl 。
该协议中使用了Bio- R ad Experion系统,但已过时了。更好的替代方法是安捷伦TapeStation系统(安捷伦,目录号:G2991AA)。该TapeStation系统可以直接测量文库池的平均DNA大小。
 



图4. Experion凝胶图像示例


MiSeq测序运行
所述MiSeq是一个集成的仪器,其执行克隆扩增,基于测序边合成,并用碱通话,对准,变体呼叫数据分析,并在单次运行报告DNA测序。在我们的方案中,我们使用MiSeq试剂盒V3(600个循环)进行测序。图URE 5示出了MiSeq试剂盒和内容通过公号码。该步骤基于MiSeq V3协议进行了修改。


 



图5. MiSeq试剂筒和孔数的内容


准备试剂盒。
从-20°C的存储中取出试剂盒。
将试剂盒放入仅包含足够室温的去离子水的水浴中,以浸没试剂盒的底部。请勿让水超过试剂盒上印制的最大水线。
让试剂盒在室温水浴中融化约60分钟或直至完全融化。
从水浴箱中取出滤筒,然后在工作台上轻轻拍打以将水从滤筒底部排出。如有必要,请干燥墨盒的底座。
翻转试剂盒十次以混合解冻的试剂,并目视检查所有位置是否解冻。
目视检查标记为IMT(掺入混合物)的物料,以确保其完全混合并且没有沉淀。
轻轻地在工作台上轻敲小柱以将水从小柱的底部排出,并减少试剂中的气泡。
注意:MiSeq吸管会移到每个板孔的底部,以吸出试剂,因此,重要的是,试剂池的底部必须没有气泡。


将试剂盒放在冰上或放在2°C至8°C的温度下,直到准备好将样品加载到试剂盒上并开始运行为止。
准备库。
变性DNA
在上样当天准备新鲜的0.2 N NaOH稀释液。为了使样品完全变性以在MiSeq上生成簇,这是必不可少的。始终从浓度为10 N或更高的NaOH浓溶液开始,否则您可能会降低簇密度。
根据Qubit浓度和平均DNA大小计算合并文库的摩尔浓度,将合并文库稀释至4 nM 。
在微量离心管中合并以下体积的样品DNA和0.2 N NaOH :
4 nM样品DNA(5 µl)
0.2 N NaOH (5微升)
短暂涡旋混合样品溶液,然后短暂离心样品溶液。
在室温下孵育五分钟以使DNA变性成单链。
将以下体积的预冷HT1加入含有变性DNA的试管中,以产生20 pM变性文库:
变性的DNA(10μl )
预冷的HT1(990μl )(在MiSeq试剂盒盒中冷冻后发货)
将变性的DNA放在冰上,直到准备进行最终稀释为止。
稀释的变性DNA
使用以下体积将变性的DNA稀释至8 pM的浓度:
20 pM DNA(400微升)
预冷HT1(600 µl)
短暂涡旋以混合DNA溶液。
脉冲离心DNA溶液。
将变性和稀释后的DNA放在冰上,直到您准备直接或在添加PhiX控件后将样品加载到MiSeq试剂盒上。
变性和语UTE PhiX控制
PhiX用作Illumina提供的内部测序对照。使用以下说明变性10nM PhiX文库并将其稀释至8 pM 。这将导致簇密度为750-850 K / mm 2 。
合并以下卷以将PhiX库稀释到2 nM :
10 nM PhiX库(2μl )
10 mM Tris- H Cl ,pH 8.5,含0.1%Tween 20(8μl )
将以下体积的2 nM PhiX文库和0.2 N NaOH在微量离心管中合并,得到1 nM PhiX文库:
2 nM PhiX库(10μl )
0.2 N NaOH (10微升)
简短地涡旋混合1 nM PhiX库解决方案。
短暂离心模板溶液。
在室温下孵育五分钟以使PhiX文库变性为单链。
将以下体积的预冷HT1加入含有变性PhiX文库的试管中,得到20 pM PhiX文库。
变性PhiX库(20微升)
预冷HT1(980μl )
如下将变性的20 pM PhiX文库稀释至8 pM :
20 pM变性PhiX文库(400μl )
预冷的HT1(600μl )
混合样品库和PhiX控件
将以下体积的变性PhiX对照文库与变性样品库结合在一起,可得到15%的体积比:
8日下午PhiX控制库(105微升)
变性样品库(595微升)
将合并的样品库和PhiX控件放在冰上,直到准备好将其加载到MiSeq试剂盒上为止。
将样品库装载到试剂盒上
使用单独的,干净且空的1 ml移液器吸头,将箔封条刺穿试剂盒上标有“装载样品”的储液罐,位置17。
移液器700微升您的样本库到加载样本库。
将旧的Nextera底漆装载到墨盒上。
Old Nextera底漆是自定义的Read 1底漆,可加到试剂盒上的位置12。表1中列出了旧的Nextera引物序列。
在新的微量离心管中,加入3.8 µl 100 µM Old Nextera底漆,并置于冰上。
使用单独的,干净且空的1 ml移液器吸头,将箔封条刺穿试剂盒上容器位置12上的箔纸密封件。
吸管大约500微升的Read 1引物混合物的出使用1ml血清移液管的墨盒。
使用旧的Nextera底漆将Read 1底漆混合物添加到试管中。
短暂涡旋混合底漆溶液,然后短暂离心分离溶液。
用1 ml移液器吸头将整个引物溶液移液回试剂盒上的位置12。
注意:请勿刺破任何其他试剂位置,当将试剂盒装载到仪器上时,它们会被刺穿。


使用MiSeq控制软件(MCS)界面直接进入运行设置步骤。表2中列出了MCS所需的索引序列。


笔记:


在Illumina实验管理器(用于创建样本表的软件)中,有一个选项可以选择“ Read 1的自定义引物”。由于我们正在将自定义引物添加到位置12,因此我们不选择该选项。
仔细检查引物序列是否正确,并将其稀释至100 µM。重要的是,定制引物必须与位置12的其他引物充分混合。我们建议将3.8 µl引物吸移到新的Eppendorf管中。然后取出位置12的内容物并将其添加到Eppendorf管中。涡旋混合均匀。然后将所有内容物吸移回试剂盒的位置12。点击墨盒以确保液体落到试管底部。
千万不要加旧NEXTERA入门到位置18至20(定制测序引物)。
NaOH的储备溶液必须为10 N或更高,这一点非常重要。有些人尝试使用5 N或2 N的库存。由于某种原因,这些较低浓度的储备液不能很好地进行变性,因此必须在将样品装入MiSeq的当天进行0.2 N的稀释。
如果变性后的20 pM PhiX文库在-15°C至-25°C下保存,则最多可保存三周。三周后,簇数趋于减少。


数据分析


使用Illumina bcl2fastq管道(v.2.20.0)进行初始数据处理。该管道将根据其条形码对序列进行多路分解。在此实验设计中,每个带条形码的文库都是从RNA样本(处理组或对照组)构建的文库。在此步骤中,仅对库条形码进行多路分解。区域和Primer ID多路分解将在以下步骤中完成。
使用TCS管道(v.1.3.8)对每个目标区域进行多路分解并创建模板共有序列。有两种处理方法。
(推荐)使用TCS Web应用程序(https://tcs-dr-dept-tcs.cloudapps.unc.edu/)自动处理原始MiSeq序列。该Web应用程序询问每个测序区域的信息,包括名称,cDNA引物序列和第一轮PCR正向引物序列。选择直接上传,UNC Longleaf序列存储库或Dropbox来上传原始MiSeq序列。处理后的序列将返回到用户提供的电子邮件地址。根据每个提交批次的文件大小,处理时间为20分钟至2小时。
按照脚本中的说明,使用TCS管道脚本(https://github.com/SwanstromLab/PID/blob/master/TCS.rb)手动处理MiSeq原始序列数据。建议在运行TC后使用TCS管道' log_mutli.rb '(https://github.com/SwanstromLab/PID/blob/master/log_multi.rb)中的文件排序和日志记录脚本对文件进行排序S管道。该脚本可以在没有附加支持的情况下使用Terminal和Ruby在Linux / MacOS平台上运行,并在安装了Terminal和Ruby的Windows平台上运行。
在TCS管道中使用“ end_join.rb ”脚本(https://github.com/SwanstromLab/PID/blob/master/end_join.rb)连接成对的末端序列。
TCS的数量是一个重要的参数,它揭示了随着实际查询的初始模板的数量而增加了测序的采样深度。
使用RUBY包' viral_seq '(1.0.8)计算每个样本的突变率。可以在TCS管道(https://github.com/SwanstromLab/PID/blob/master/mut_table.rb)中找到可执行脚本。该脚本将每个单独的序列与样本共有序列进行比较,并生成一个表格,列出每种替换类型的频率。


笔记:


TCS管道的较新版本(版本≥2.0.0 )作为可执行的命令行工具包含在RUBY软件包“ viral_seq ”(版本≥1.0.8 )中。它具有几个主要更新,包括末端连接功能以及TCS的创建,因此建议将来使用。
作者致力于改善生物信息学分析流程。如有任何相关问题,欢迎用户与作者联系。


致谢


我们想感谢以下资金来源:授予TPS和RSB的美国国家过敏和传染病研究所R01 AI132178 。和NIAID 5R01 AI140970授予RS。我们感谢UNC高通量测序设施为开发此协议所提供的帮助。该协议已用于我们先前发布的工作中(Sheahan等,2020)。


利益争夺


北卡罗莱纳大学正在寻求对Primer ID的IP保护,RS已获得象征性的特许权使用费。


参考


亚伯拉罕,MR,约瑟夫,SB,加勒特,N.,泰尔斯,L.,Moeser ,M.,Archin ,N.,理事会,OD,Matten ,D.,周,S.,Doolabh ,D.,安东尼,C 。,Goonetilleke ,N.,Karim,SA,Margolis,DM,Pond,SK,Williamson,C.,and Swanstrom ,R.(2019)。具有复制能力的HIV-1潜在贮库主要是在治疗开始时建立的。Sci Transl Med 11(513):eaaw5589。
Adewumi ,OM,Dukhovlinova ,E.,Shehu ,NY ,Shou ,S。,理事会,OD,Akanbi ,MO,Taiwo ,B.,Ogunniyi ,A.,Robertson,K.,Kanyama ,C.,Hosseinipour ,MC和Swanstrom ,R.(2020年)。在尼日利亚和马拉维的非B型HIV-1感染中,HIV-1中枢神经系统分隔和细胞因子相互作用。AIDS Res Hum Retroviruses 36(6):490-500。
杂波,DS,周,S.,Varghese表示,五,李承晚,SY斯基,BA,杰弗里Fessel ,W.,克莱恩,DB,Spielvogel ,E.,福尔摩斯,SP,赫尔利,LB,西尔弗伯格,MJ,Swanstrom ,R.和Shafer,RW(2017)。通过Sanger测序检测未治疗的HIV-1感染个体中耐药耐药少数民族变异的普遍性,这些个体有和没有传播耐药性。感染杂志216(3):387-391。
理事会,OD,Zhou,S.,McCann,CD,Hoffman,I.,Tegha ,G.,Kamwendo ,D.,Matoga ,M.,Kosakovsky Pond,SL,Cohen,MS和Swanstrom ,R。(2020)。深度测序揭示了有或没有与性传播感染相关的尿道炎的男性男性精液中的房室HIV-1。J Virol 94(12):e00151-20。
Dennis,AM,Zhou,S.,Sellers,CJ,Learner,E.,Potempa ,M.,Cohen,MS,Miller,WC,Eron ,JJ和Swanstrom ,R.(2018)。使用Primer-ID深度测序来检测最近的人类免疫缺陷病毒1型感染。Ĵ传染病杂志218(11):1777至1782年。
Jabara ,CB,Jones,CD,Roach,J.,Anderson,JA和Swanstrom ,R。(2011)。使用引物ID对HIV-1蛋白酶基因进行准确采样和深度测序。PROC国家科科学院科学美国阿108(50):20166-20171。
Keys,JR,Zhou,S.,Anderson,JA,Eron ,JJ,Jr.,Rackoff ,LA,Jabara ,C. and Swanstrom ,R.(2015年)。引物ID通知下一代测序平台并揭示HIV-1逆转录酶编码域中先前存在的耐药突变。AIDS Res Hum逆转录病毒31(6):658-668。
Lee,SK,Zhou,S.,Baldoni ,PL,Spielvogel ,E.,Archin ,NM,Hudgens ,MG,Margolis,DM and Swanstrom ,R.(2017年)。使用超深度测序和引物ID在病毒生长分析中对潜在的HIV-1储库进行定量。Ĵ Acquir免疫Defic Syndr 74(2):221-228。
刘易斯,CA,Jr。的Crayle ,J.,周,S.,Swanstrom ,R。和沃尔芬登,R。(2016)。在地球历史上,胞嘧啶脱氨和自发突变的急剧下降。PROC国家科科学院科学美国阿113(29):8194-8199。
Liu L.,Li,Y.,Li,S.,Hu,N.,He,Y.,Pong,R.,Lin D.,Lu,L. and Law,M.(2012)。下一代测序系统的比较。生物技术杂志(J Biomed Biotechnol)2012:251364。
Sheahan ,TP,Sims,AC,Zhou,S.,Graham,RL,Pruijssers ,AJ,Agostini ,ML,Leist ,SR,Schafer,A.,Dinnon ,KH,3rd,Stevens,LJ,Chappell,JD,Lu, X.,休斯,TM,乔治,AS,希尔,CS,蒙哥马利,SA,布朗,AJ,Bluemling ,GR,Natchus ,MG,萨丹丹,M.,科利卡洛夫,AA,画家,G.,哈科特,J.,达明,A.,索恩伯格,NJ,Swanstrom ,R.,丹尼森,MR和巴里奇,RS(2020)。口服可生物利用的广谱抗病毒剂可抑制人呼吸道上皮细胞培养物中的SARS-CoV-2和小鼠中的多种冠状病毒。Sci Transl Med 12(541):eabb5883。
叶,J.,Coulouris ,G.,Zaretskaya ,I.,Cutcutache ,I.,蔷薇,S.和Madden,TL(2012)。Primer-BLAST:设计用于聚合酶链式反应的靶标特异性引物的工具。BMC生物信息学13:134。
Zhou,S.,Bednar ,MM,Sturdevant ,CB,Hauser,BM和Swanstrom ,R.(2016)。HIV-1 env基因的深度测序揭示了V1 / V2和V3可变区中离散的X4谱系和X4和R5病毒之间的连锁不平衡。Ĵ病毒学杂志90(16):7142-7158。
周,S.,琼斯,C.,Mieczkowski ,P。和Swanstrom ,R。(2015)。引物ID验证了模板的采样深度,并大大降低了HIV-1基因组RNA种群的下一代测序的错误率。J Virol 89(16):8540-8555。
周,S.,Williford ,SE,McGivern ,DR,伯奇,CL,胡,F.,苯(C6H6),T.,Ingravallo ,P.,Asante-阿皮亚,E.,豪,AYM,Swanstrom ,R。和柠檬, SM(2018)。基因1型丙型肝炎病毒中NS5A抑制剂耐药性的进化途径。Antiviral Res 158:45-51。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhou, S., Hill, C. S., Clark, M. U., Sheahan, T. P., Baric, R. and Swanstrom, R. (2021). Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome . Bio-protocol 11(5): e3938. DOI: 10.21769/BioProtoc.3938.
  2. Sheahan, T. P., Sims, A. C., Zhou, S., Graham, R. L., Pruijssers, A. J., Agostini, M. L., Leist, S. R., Schafer, A., Dinnon, K. H., 3rd, Stevens, L. J., Chappell, J. D., Lu, X., Hughes, T. M., George, A. S., Hill, C. S., Montgomery, S. A., Brown, A. J., Bluemling, G. R., Natchus, M. G., Saindane, M., Kolykhalov, A. A., Painter, G., Harcourt, J., Tamin, A., Thornburg, N. J., Swanstrom, R., Denison, M. R., and Baric, R. S. (2020). An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med 12(541): eabb5883.
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