Protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach

Summary

Lumpy skin disease (LSD) is a viral disease predominantly affecting cattle caused by a poxvirus belonging to the capripoxvirus genus. Here, we present a protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach. We describe steps for DNA extraction, viral DNA enrichment, amplicon pooling and purification, and library preparation and pooling. We then detail procedures for sequencing and computational analysis. This protocol can be adapted to any Illumina sequencing platform as an accelerated and scalable system.

For complete details on the use and execution of this protocol, please refer to Bhatt et al.^1,2

Graphical abstract

Highlights

• •Rapid and scalable approach for detection and whole-genome sequencing of LSDV
• •Uses multiplex-primer pools for viral nucleic acid enrichment
• •Compatible with any Illumina sequencing platforms
• •Rapid analysis with automated open-source toolkit “genepi-boxpox”

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Lumpy skin disease (LSD) is a viral disease predominantly affecting cattle caused by a poxvirus belonging to the capripoxvirus genus. Here, we present a protocol for next-generation sequencing of the LSD virus genome using an amplicon-based approach. We describe steps for DNA extraction, viral DNA enrichment, amplicon pooling and purification, and library preparation and pooling. We then detail procedures for sequencing and computational analysis. This protocol can be adapted to any Illumina sequencing platform as an accelerated and scalable system.

Before you begin

Introduction

Lumpy Skin Disease is a disease of cattle with high morbidity and mortality caused by the Lumpy Skin Disease Virus (LSDV) LSDV is a double-stranded DNA virus of ∼151 kbp size genome and belongs to the genus capripoxvirus of the poxviridae family of viruses. Lumpy Skin Disease in cattle is characterized by fever, and lymphadenopathy along with the appearance of multiple nodules on the skin and mucous membranes, giving its name. The disease often results in poor growth of cattle, a reduction in milk production, abortion or infertility, and in severe cases death of the affected animal.³ By impacting animal health, LSD thus has socio-economic effects relating to the trade of the animals and their by-products. LSD is listed as an emerging transboundary notifiable viral disease that severely affects livestock economics by the World Organisation for Animal Health (OIE).⁴

While the disease has been endemic in Africa, the previous decade has seen significant outbreaks in Asia. These outbreaks have provided interesting genomic insights.^5,6,7 Our report on LSDV isolates from India suggests that the viral genomes from the 2022 outbreak form a distinct lineage and harbor a large number of genetic mutations when compared to the LSDV reference genome.² The report further suggests the importance of genomic surveillance for developing mitigation strategies for disease outbreaks including understanding viral evolution and development of diagnostic tools. The present genomic efforts have been reliant on metagenomic approaches for sequencing, which limits the scale and wider applicability of genomic surveillance. Amplicon-based targeted approaches have extensively been used for genomic surveillance and epidemiology of SARS-CoV-2.⁸ We surmise that such an approach could be adapted for LSDV. This protocol uses a unique set of primers designed for the targeted enrichment of the LSDV genome using the multiplex PCR approach. The sequencing-ready libraries were synthesized using the Illumina COVIDSeq test (RUO) with the marginal modification in the original protocol.¹ To aid researchers with bioinformatics analysis of the generated LSDV sequencing data, we have developed an automated toolkit Genetic Epidemiology in a Box for Poxviruses (genepi-boxpox) for assembly and analysis of the genomic data which can be implemented without the requirement for dedicated and expert bioinformatics resources. The open-source toolkit is available as a GitHub repository⁹ for download and use.

This protocol has been standardized to be executed using the MiSeq sequencing platform by Illumina. However, considering the number of samples and the throughput required this protocol can be adapted to any Illumina sequencers, therefore could serve as a single assay that can be scaled for quick detection as well as genetic epidemiology of LSDV.

This protocol consists of the following steps:

DNA extraction- DNA is extracted from the inactivated specimens collected from the animal in viral transport media (VTM).

Primers Designing and Pooling- LSDV-specific multiplex PCR primer designing and primer pooling.

Target amplification- To enrich the viral genome present in the sample, the isolated DNA will undergo three separate PCR reactions using the primer that selectively enriches the LSDV genome in the sample.

Amplicon pooling- The PCR amplified products (Pool 1, 2 and 3) from the previous step will be pooled in a single tube.

Library preparation, Library Pooling, and Quantification- The pooled and amplified PCR products are fragmented followed by tagging to adapter sequences (tagmentation). Another round of PCR is run for the adapter-tagged fragments post which the indexed tagged libraries are further pooled and cleaned using purification beads. Qubit High Sensitivity dsDNA quantification kit (Invitrogen Life technologies, USA) is used to quantify the pooled library product.

Sequencing- After the quality check, the sequencing-ready libraries are sequenced on the MiSeq platform by Illumina using the sequencing by synthesis (SBS) chemistry.

Analysis- The raw sequencing data generated is demultiplexed into individual FASTQ files, genome assembly and variant calling for each sequenced sample are performed using the genepi-boxpox tool.

DNA extraction

Timing: [1 h]

During this process, DNA would be extracted from the specimen collected from the infected animal. The nucleic acid extraction uses Nextractor NX-48S as instructed by the manufacturers (Genolution Inc. Korea).

(All steps should be carried out within the BSL-2 category Biosafety cabinets).

• 1.
  Specimen heat inactivation.

  • a.
    Vortex the VTM vials briefly.
  • b.
    Transfer around 500 μL of the VTM containing the specimen collected from the animal to 1.5 mL DNase/RNase free microcentrifuge tube (MCT).
  • c.
    Keep the vial containing the specimen on a heat block with the temperature adjusted to 50°C for 15 min. Aliquot 200 μL of the heat-inactivated VTM in the cartridge provided with the extraction kit and select the program for the DNA extraction as per the instructions provided in the manual (Nextractor NX-48S).
  • d.
    Transfer the extracted DNA samples from the cartridge to the new 1.5 mL MCT vials.

Pause point: The isolated DNA samples may be stored at −20°C until required for further use.

Multiplex primer design

In this step, a predesigned set of LSDV-specific multiplex primers would be used. The primers are pre-designed using PrimalScheme¹⁰ and manually checked for their target specificity and primer-dimer formation. The designed primers were synthesized by a primer synthesis service provider (Eurofins Genomics, India).

• 2.
  Primer dilution and pooling.

  • a.
    The primer set includes LSDV-specific overlapping multiplex PCR primers with an approximate 5 kb of the amplicon size. The list of primer sequences and their genomic loci is summarized in Table S1.¹¹
  • b.
    The obtained primers can be diluted to 100 μM concentration with Nuclease-Free Water (NFW).
  • c.
    Diluted primers can be pooled in the respective primer pools (Table S1) so that the concentration of each primer in the pool will become 200 nM.
  • d.
    The above combination of the LSDV primers will result in three individual multiplex PCR primer pools that can be further used in the PCR reaction.

Pause point: The LSDV primer pools can be stored at −20°C till further use.

Target amplification

Timing: [3 h]

In this step, the LSDV viral genome in the specimen will be selectively enriched using the three primer pools containing the primers which specifically amplify the LSDV genomes.

• 3.
  Amplification of the LSD viral genome.

  • a.
    Label three 8-strip PCR tubes as LSDV_P1, LSDV_P2, and LSDV_P3.
  • b.
    Prepare LSDV_P1, LSDV_P2, and LSDV_P3 mastermix in three separate 1.5 mL MCT vials using TAKARA PrimeStar GXL kit (Takara Bio Inc. Japan) as mentioned in the table below.
  • c.
    Pipette mix the content in the tube properly and spin the tube briefly.
  • d.
    Aliquot 5 μL of the LSDV isolated DNA samples in each strip tube marked as LSDV_P1, LSDV_P2, and LSDV_P3.
  • e.
    Add 45 μL of the reaction master mix containing an individual LSDV multiplex primer pool (LSDV_Pool1/ LSDV_Pool2 / LSDV_Pool3).
  • f.
    Pipette mix the content in the strip tube and spin at 1000 × g for 30 s.
  • g.
    Choose the preheat lid option in a thermal cycler and set the lid temperature as 105°C.
  • h.
    Set the final reaction volume as 50 μL.
  • i.
    Run the following program after placing the 8 strip tubes on the thermal cycler.
  • j.
    PCR cycling conditions.

Step	Temperature	Time	Cycles
Denaturation	98°C	10 s	30
Annealing	60°C	15 s
Extension	68°C	5 min
Hold	4°C	Ongoing

Under current standard cycling conditions, the typical run takes approximately 3 h 05 min.

Reagent	Volume (for 50 μL reaction)
5X PrimeSTAR GXL Buffer	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Primer Pool (LSDV_Pool1/LSDV_Pool2/ LSDV_Pool3)	3 μL
PrimeSTAR GXL DNA Polymerase	1 μL
Nuclease Free Water (NFW)	to 50 μL

Pause point: The protocol can be paused here and the PCR amplified samples can be stored at −20°C for up to 3 days.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Nextractor NX-48S	Genolution	NX-48S
COVIDSeq assay (96 Samples) or (3,072 samples) test kit	Illumina Inc.	20043675/ 20049393
IDT for Illumina PCR indexes set 1–4 (384 indexes)	Illumina Inc.	20043137
MiSeq reagent kit v3 (150-cycle)	Illumina Inc.	MS-102-3001
Qubit dsDNA HS assay kit	Thermo Fisher Scientific	Q32851
Molecular biology grade ethanol	Honeywell International Inc.	32221
PrimeSTAR GXL DNA polymerase	Takara Bio Inc.	R050A
Microfuge tubes (MCTs)	Thermo Fisher Scientific	AM12400
Conical tubes, 15 mL	Eppendorf	0030122151
Thermal cycler (PCR).	Thermo Fisher Scientific	NA
Thermo mixer with a 96-well PCR plate adapter.	Eppendorf	EP5382000031
Single/multichannel pipette sets	Eppendorf/Thermo Fisher Scientific/any other	NA
DynaMag-96 side magnet	Thermo Fisher Scientific	12331D
DynaMag-2 magnet	Thermo Fisher Scientific	12321D
Qubit 4 fluorometer	Thermo Fisher Scientific	Q33238

Materials and equipment

Reagent	Description	Storage	Total volume required per reaction
LSDV DNA Sample∗	Sample	−20°C	5 μL
NX-48S Viral NA Kit∗	DNA/RNA extraction cartridge	Room temperature (RT)	1 cartridge for 24 samples
LSDV_PP1∗	LSDV Primer Pool 1	−20°C	3 μL
LSDV_PP2∗	LSDV Primer Pool 2	−20°C	3 μL
LSDV_PP3∗	LSDV Primer Pool 3	−20°C	3 μL
ELB HT	Elution Buffer HT	−20°C	8.5 μL
EPH3 HT	Elution Prime Fragment 3HC Mix	−20°C	8.5 μL
IPM HT	Illumina PCR Mix HT	−20°C	15 μL
TB1 HT	Tagmentation Buffer 1 HT	4°C	12 μL
EBLTS HT	Enrichment BLT HT	−20°C	4 μL
ST2 HT	Stop Tagment Buffer2 HT	4°C	10 μL
TWB HT	Tagmentation Wash Buffer HT	4°C	200 μL
EPM HT	Enhanced PCR Mix HT	−20°C	24 μL
Index adapter∗	IDT for Illumina PCR Indexes- Set 1,2,3,4	−20°C	10 μL
ITB	Illumina Tune Beads	RT	396 μL (for 96 samples)
RSB	Resuspension Buffer	4°C	55 μL
NFW∗	Nuclease Free Water	RT	25 mL

∗- Items not included along with the Illumina COVIDSeq test kit.

Step-by-step method details

Sequencing-ready library preparation and library pooling

Timing: [3–4 h]

In this step, libraries for sequencing would be prepared from the LSDV amplicons after tagmentation, indexing and further amplification by PCR.

• 1.
  Tagmentation of the PCR-amplified LSDV product.

  • a.
    Pool 10 μL of the PCR amplified product from each well of LSDV_P1, LSDV_P2, and LSDV_P3 PCR strip tubes in the respective well of the new PCR strip tube, and pipette mix the content in the tubes. This will result in a total volume of 30 μL in each of the PCR strip tube’s individual wells.
  • b.
    Label the new 8-strip PCR tube as LSDV TAG tubes.
  • c.
    Transfer 20 μL of the pooled amplicons from previous PCR tubes in respective wells of the new PCR strip tube (TAG Tube).
  • d.
    Make the Tagmentation mix as follows;

    • i.
      Take a 15 mL conical centrifuge tube and label it as Tagmentation Mastermix.
    • ii.
      Add 1920 μL (20 μL × 96 samples) of NFW.
    • iii.
      Add 1152 μL of TB1 HT (12 μL × 96 samples).
    • iv.
      Add 384 μL of EBLTS HT (4 μL × 96 samples).
    • v.
      To mix the contents in the tube, vortex it briefly.
  • e.
    To each individual well of the PCR strip tube add 30 μL of the tagmentation mix.
  • f.
    Pipette mix the content in the tube and centrifuge the PCR strip tubes for 30 s at 1000 × g at RT.
  • g.
    Set the lid temperature at 100°C after choosing the preheat lid option.
  • h.
    Set the total reaction volume as 50 μL.
  • i.
    Run the following program after placing the strip tube (TAG tubes) in a thermal cycler.
  • j.
    PCR cycling conditions.

Step	Temperature	Time
Tagmentation	55°C	5 min
Hold	10°C	ongoing

• 2.
  Clean-up (Post tagmentation).

  • a.
    Centrifuge the LSDV TAG tube at 500 × g for 1 min.
  • b.
    Add 10 μL of ST2 HT to each well of the LSDV TAG tube.
  • c.
    Pipette mix the content in the tube.
  • d.
    Incubate LSDV TAG tubes at room temperature for 5 min.
  • e.
    The LSDV TAG tubes should be centrifuged at 500 × g for 1 min.
  • f.
    Place the tubes on the magnetic stand after removing their caps.
  • g.
    Wait for 3 min or until the liquid is clear.
  • h.
    Without disturbing the beads affixed to the tube walls, remove and discard all supernatant from the wells.
  • i.
    Wash the beads as per the following steps;

    • i.
      Remove the LSDV TAG tubes from the magnetic stand.
    • ii.
      add 100 μL of TWB HT to each well.
    • iii.
      Mix the contents in the tube using a pipette, close the caps of the tubes, and centrifuge at 500 × g for 1 min.
    • iv.
      Remove the cap and place the strip tube on the magnetic stand. Keep for 3 min or until the liquid is clear.
    • v.
      Remove and discard the supernatant from each well.
    • vi.
      Repeat step i (wash steps) one more time.

CRITICAL: To prevent the over-drying of beads, do not discard the supernatant after the second wash.

• 3.
  PCR amplification of the tagmented product.

  • a.
    Prepare the Enhanced PCR Mix as described in the following steps;

    • i.
      Add 2304 μL (24 μL × 96 samples) of EPM HT.
    • ii.
      Add 2304 μL (24 μL × 96 samples) of Nuclease-free water.
    • iii.
      To mix the contents, vortex the tube briefly.
  • b.
    The supernatant from the LSDV TAG tubes should be removed and discarded.
  • c.
    Use a 10 μL or 20 μL pipette to remove any remaining TWB HT from the LSDV TAG tubes.
  • d.
    To each well of the LSDV TAG tubes add 40 μL of Enhanced PCR Mix.
  • e.
    To each well of the LSDV TAG tubes add 10 μL of index adapters.
  • f.
    Mix the contents of the tubes using a pipette and centrifuge the tube at 500 × g for 1 min.

    CRITICAL: Make sure the beads are entirely resuspended by inspecting the tubes.

  • g.
    Set the temperature to 100°C after choosing the preheat lid option.
  • h.
    Set the total volume of the reaction to 50 μL.
  • i.
    Run the PCR program as follows, and after placing the LSDV TAG tubes on a thermal cycler.
  • j.
    PCR cycling conditions:

Step	Temperature	Time	Cycles
Pre-amp	72°C	3 min	1
Initial Denaturation	98°C	3 min	1
Denaturation	98°C	20 s	7
Annealing	60°C	30 s
Extension	72°C	1 min
Final Extension	72°C	3 min	1
Hold	10°C	Ongoing

• 4.
  Library pooling and clean-up.

  • a.
    Centrifuge the LSDV TAG tubes at 500 × g for 1 min at RT.
  • b.
    Place the tubes on a magnetic stand after opening the caps. Keep it on the stand for 3–5 min or until the liquid is clear.
  • c.
    Transfer 45 μL of the clear supernatant from each LSDV TAG tube (from the previous step) to fresh 1.5 mL MCT vials. This step will result in the 8 individual 1.5 mL tubes.

    CRITICAL: Do not disturb the beads attached to the wall of the tubes while transferring the content.

  • d.
    To each tube, add 40.9 μL of ITB (ITB volume is calculated as 0.9 times the sum of the libraries' volume).
  • e.
    To mix the contents, vortex the tube.
  • f.
    Incubate the tube for 5 min at room temperature.
  • g.
    Briefly spin the tube and place it on a 1.5 mL magnetic stand. Keep on the stand for 5 min.
  • h.
    Remove and discard all supernatant in the tube.
  • i.
    Wash the beads with 80% ethanol by adding 1000 μL of freshly prepared 80% ethanol to the tube.
  • j.
    Wait for 30 s.
  • k.
    Remove the supernatant and discard it.
  • l.
    Repeat the wash steps once again. A total of 2 washes with 80% ethanol should be done.
  • m.
    Remove any remaining ethanol left in the tube.
  • n.
    Air-dry the beads for 2 min.
  • o.
    Add 55 μL of Resuspension Buffer to the tube.
  • p.
    Vortex the tube to mix the contents.
  • q.
    Incubate the tube for 2 min at room temperature.
  • r.
    Spin the tube briefly, place it in the magnetic stand, and then keep it standing for 2 min.
  • s.
    Take a new 1.5 mL tube and label it as “Final library”.
  • t.
    Transfer 50 μL of the supernatant into the tube labeled as Final library.

    Pause point: The protocol can be paused here.

    The Final library tubes may be stored at −20°C for at least 7 days.
• 5.
  Quantification and Normalization of the synthesized library.

  • a.
    To quantify the synthesized libraries, the protocol uses the Qubit High Sensitivity dsDNA quantification kit (Invitrogen Life technologies, USA). To check the size of the library, load 2 μL of the library onto a 2% agarose gel. The libraries should be observed as a band at a size around 300–400 bp corresponding to the sizing ladder (Figure 1).
  • b.
    To pool the synthesized libraries at 4 nM concentration use the Illumina pooling calculator (https://support.illumina.com/help/pooling-calculator/pooling-calculator.htm) applying the following settings.
  • c.
    Illumina pooling calculator settings.

Description	Unit
Library Plexity	8
Unit of Measure for Library	ng/μL
Library Size	300 bp
Pooled Library Concentration (nM)	4 nM
Total Pooled Library Volume (μL)	25 μL

Figure 1.

The quality of the sequencing-ready libraries was confirmed by agarose gel electrophoresis (2%), and the expected band of ∼300 bp size was detected on the gel (L- 100 bp DNA ruler, S1-S8- sequencing-ready libraries)

Sequencing the libraries

Timing: [∼22 h: for MiSeq reagent kit V3 (2 × 75 bp)]

[The prepared libraries for Lumpy Skin Disease Virus are sequenced on the MiSeq sequencing platform by Illumina].

• 6.
  Library dilutions and sequencing run set-up.

  • a.
    Thaw the MiSeq sequencing reagents by keeping the reagent cartridge in the water bath at RT for 15–30 min, after complete thawing, the thawed cartridge can be kept at 4°C till further use.
  • b.
    Preparing PhiX (Optional).

    • i.
      Take a fresh 1.5 mL MCT tube and label it as PhiX Control.
    • ii.
      To make a 4 nM PhiX library, add 2 μL of PhiX stock library (10 nM) and 3 μL of Nuclease-free water.
    • iii.
      Take a new 1.5 mL tube and label it as PhiX-Final. Add 5 μL of diluted PhiX (4 nM) and 5 μL of 0.2 N freshly prepared NaOH to the PhiX-Final tube.
    • iv.
      To mix the contents of the tube, vortex it briefly. Spin the tube and incubate it for 5 min at room temperature.
    • v.
      To the PhiX-Final tube, add 990 μL of prechilled HT1. This will result in 1 mL of 20 pM PhiX library.
  • c.
    Denaturation of final LSDV library.

    • i.
      Take a 1.5 mL tube and label it as a 20 pM library. Add 5 μL of the diluted library (4 nM) and 5 μL of 0.2N freshly prepared NaOH to the tube.
    • ii.
      To mix the contents of the tube, vortex it briefly. Spin the tube and incubate it for 5 min at room temperature.
    • iii.
      To the 20 pM library tube add 990 μL of prechilled HT1. This will result in 1 mL of 20 pM Final library.
    • iv.
      Take a new 1.5 mL tube and label it as 7 pM Final Library tube.
    • v.
      Aliquot 210 μL of volume from the 20 pM library tube.
    • vi.
      Add 390 μL of prechilled HT1 and mix well.
    • vii.
      From the 7 pM tube remove and discard 6 μL of the mixture.
    • viii.
      Add 6 μL of the denatured 20 pM PhiX library to the 7 pM denatured library tube for 1% spike-in (optional).
    • ix.
      Use a pipette to mix the contents of the tube. Spin the tube briefly and place it on ice. The denatured library can now be loaded into the flow cell.

CRITICAL: Please note that the final library must be loaded onto the flow cell within 30 min of preparation.

Note: Denaturation of the sequencing-ready library and PhiX can be done simultaneously to speed up handling.

Expected outcomes

The final library will appear as approximately a 300 bp-sized band on 2% agarose gel (Figure 1). Error in tagmentation or size selection will result in deviation in the fragment size on the gel, which can be minimized by adopting good laboratory practices and proper pipetting practices.

Quantification and bioinformatics analysis

• 1.Genome Assembly and Analysis.
  This section summarizes the computational analysis steps and pipeline for assembling LSDV genomes from the raw sequence data generated and calling genetic variants in the assembled genomes. The steps include command line options in Linux.

  • a.
    Raw sequence data that will be generated by the Illumina MiSeq platform will be in the form of Binary Base Call (BCL) files. Before further processing and analysis, the BCL files need to be converted to FASTQ format before further processing. The bcl2fastq conversion software, an open-source tool provided by Illumina, can be used to demultiplex BCL files to FASTQ files using the following command:
    bcl2fastq --runfolder-dir <path to the folder containing sequencing run data> --sample-sheet <path to the sample sheet file containing the sample and index details> --output-dir <name of the folder where the demultiplexed output FASTQ files are to be stored>
  • b.
    Analysis for all sequenced samples can be performed using the genepi-boxpox tool which automates the task of assembling genomes from raw FASTQ files. The tool can be installed following the installation instructions given on the GitHub page.⁹ After successful installation, the tool opens locally as a web page on the user’s system where the user may upload all necessary input files.
  • c.
    The genepi-boxpox tool takes in as input raw data generated by the sequencer, the sample sheet used to demultiplex the BCL files, and a metadata file containing details about the samples that have been sequenced. Details about the input files required to run the tool can be found in the GitHub repository of the tool.⁹
  • d.
    The genepi-boxpox tool automates the genome assembly pipeline for the samples that have been provided as input. The steps that are followed for genome assembly include performing quality control of the raw FASTQ files using FastQC and Trimmomatic (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), mapping quality-controlled files against the LSDV reference genome (NC_003027.1)¹² to generate a reference-based assembly for the sequenced samples using HISAT2,¹³ variant calling and consensus FASTA generation using VarScan,¹⁴ SAMtools¹⁵ and seqtk (https://github.com/lh3/seqtk).
  • e.
    The tool will output a summary report for the analysis, including the detected mutations in each sample along with the coverage statistics of each genome assembly.
  • f.
    The FASTA sequence files generated by genepi-boxpox tool can also be used to understand evolutionary relationships between the sequenced samples and other publicly available LSDV genomes. Open-source toolkits such as Nextstrain¹⁶ can be used for performing phylogenetic analysis of the samples.
    An example of the different sequencing and analysis metrics that can be used to assess the overall quality of the assembled genomes for one of the sequenced samples is given in Table 1.

Table 1.

An example of the different sequencing and analysis metrics

Sample ID	Number of raw reads R1	Number of raw reads R2	Number of reads after trimming R1	Number of reads after trimming R2	% Of reads aligned with reference	Total base pairs that mapped to reference	X coverage	Genome coverage	Number of variants called	Time taken
LSDV_01	1312536	1312536	1217200	1217200	91.7	148559700	1097.78	99.96	175	378 s

Limitations

An amplicon-based enrichment method for sequencing can be affected by several factors such as Ct value of the collected specimens, an enzyme used for the target enrichment PCR. Also, the limitations related to the target amplification can be minimized by using high-fidelity Taq polymerase enzymes. The methodology for computational assembly and analysis of the raw sequencing data does not consider potential inaccuracies in intra-host viral variant frequency caused by mismatched primers. Interpreting variants identified within amplicons containing mismatches in the primer binding regions should be done cautiously.

Troubleshooting

Problem 1

DNA extraction- unavailability of the automated DNA extraction system (related to step: DNA extraction).

Potential solution

The current protocol is standardized on the LSDV genomic DNA isolated using Nextractor, an automated DNA/ RNA extraction platform. In case of the availability of the same users can adapt the other bead-based automated nucleic acid extraction platform or column-based manual viral nucleic acid extraction kits such as QIAamp Viral RNA Mini Kit (QIAGEN, part # 52906).

Problem 2

Target Amplification- there is no evidence of PCR amplification (related to step: Target Amplification).

Potential solution

Check the quality and integrity of the PCR reagents, including the template DNA, primer pools, nucleotides, buffer, and polymerase. It is important to ensure that the reagents have not expired and have been stored and handled properly. Also verify the correct assembly of the PCR reaction, including proper pipetting of reagents, avoiding cross-contamination, and using appropriate controls. Double-checking the volumes and concentrations of the components may help identify any mistakes or discrepancies. The recommended concentration of template (PCR amplified product) for the library preparation ranges from 1–500 ng. Check the concentration of the PCR amplified purified product in case the troubleshooting is required.

Problem 3

No expected band visible during the library quality check (related to step: Sequencing-ready Library Preparation and Library Pooling).

Potential solution

Carefully review the experimental protocol and ensure that all steps were followed correctly. This includes verifying the library preparation protocol, tagmentation, adapter ligation, and PCR amplification conditions. Verify the size distribution of the DNA fragments using an appropriate method such as agarose gel electrophoresis or capillary electrophoresis. Compare the observed fragment sizes with the expected size range for the library. If the observed fragment sizes are significantly different from the expected range, it may indicate issues with tagmentation or size selection steps.

Problem 4

The Amplicon Sequencing experiment fails to generate near complete genome (related to step: Genome Assembly and Analysis).

Potential solution

Ensure that the viral DNA used for the Amplicon Sequencing experiment is of high quality and sufficient quantity. Assess the library preparation steps, including amplicon tagmentation, adapter ligation, and library amplification.

Problem 5

Low number of reads remaining in the FASTQ file after quality control (related to step: Genome Assembly and Analysis).

Potential solution

The current genome assembly pipeline will trim bases from the raw FASTQ files that have a Phred quality score less than Q30. In case a majority of reads are being dropped after trimming the FASTQ files, users may check the FastQC reports generated for the sample to check parameters such as per base sequence quality and per sequence quality scores to ensure that good quality data has been generated in the sequencing run.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Vinod Scaria (vinods@igib.in) and Dr. Sridhar Sivasubbu (sridhar@igib.in).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Dr. Vinod Scaria, (vinods@igib.in).

Materials availability

Detailed information about the reagents, kits and consumables used in the protocol is given in the “key resources table”.

Data and code availability

The genepi-boxpox tool used for automated genome assembly and analysis is available as a GitHub repository⁹ for download and use.

Supplemental information

Table S1. Details of the LSDV multiplex primers and pooling guide, (related to Before You Begin Step 2c)

https://docs.google.com/spreadsheets/d/13Q-hwF1mE0VdqPH711k1f1Vt_BUWjOHljmLW-6tAwJQ/edit?usp=sharing.