Sequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1

Sara França de Araújo dos Santos; Ueric José Borges de Souza; Martha Trindade Oliveira; Jairo Jaime; Fernando Rosado Spilki; Ana Cláudia Franco; Paulo Michel Roehe; Fabrício Souza Campos

Jul 14, 2023

Sequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1

DOI

dx.doi.org/10.17504/protocols.io.36wgq3563lk5/v1

Sara França de Araújo dos Santos¹,
Ueric José Borges de Souza¹,
Martha Trindade Oliveira²,
Jairo Jaime³,
Fernando Rosado Spilki⁴,
Ana Cláudia Franco²,
Paulo Michel Roehe²,
Fabrício Souza Campos²

¹- Bioinformatics and Biotechnology Laboratory, Campus of Gurupi, Federal University of Tocantins, Gurupi 77410-570, Brazil;
²Virology Laboratory, Department of Microbiology, Immunology, and Parasitology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre 90035-003, Brazil;
³Universidad Nacional de Colombia, Sede Bogotá. Facultad de Medicina Veterinaria y de Zootecnia, Departamento de Salud Animal. Centro de Investigación en Infectología e Inmunología Veterinaria (CI3V). Carrera 30 # 45-03, Bogotá D.C. CP 11132. Colombia;
⁴Molecular Microbiology Laboratory, Feevale University, Novo Hamburgo 93525-075, Brazil

Martha Oliveira

UFRGS

DOI: dx.doi.org/10.17504/protocols.io.36wgq3563lk5/v1

Protocol Citation: Sara França de Araújo dos Santos, Ueric José Borges de Souza, Martha Trindade Oliveira, Jairo Jaime, Fernando Rosado Spilki, Ana Cláudia Franco, Paulo Michel Roehe, Fabrício Souza Campos 2023. Sequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3563lk5/v1

Manuscript citation:

Santos SFA, Souza UJB, Oliveira MT, Jaime J, Spilki RS, Franco AC, Roehe PM, Campos FS. Recovery of complete genomes of canine parvovirus from clinical samples. bioRxiv preprint, 25p. 2023.https://www.biorxiv.org/content/10.1101/2023.07.12.548703v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: June 30, 2023

Last Modified: July 14, 2023

Protocol Integer ID: 84318

Keywords: CPV, Canine Parvovirus, sequencing, viral DNA, rectal swab, DNA extraction

Funders Acknowledgement:

This work was partially supported by grants from Rede Corona-ômica BR MCTI/FINEP (http://www.corona-omica.br-mcti.lncc.br, accessed on 25 June 2023) affiliated by RedeVírus/MCTI

Grant ID: FINEP 01.20.0029.000462/20; CNPq 404096/2020-4

This work was partially supported by grant from the National Council for Scientific and Technological Development–CNPq

Grant ID: Process number: 443215/2019-7

Abstract

Canine parvovirus (CPV) is a highly contagious viral disease that affects dogs, especially puppies. CPV-2 is recognized for its resilience in contaminated environments, ease of transmission among dogs, and pathogenicity for puppies. In this protocol, we have adapted the methodology to allow the recovery of complete CPV-2 genomes directly from clinical samples (dry swabs) from puppies with clinical signals of viral enteritis. A multiplex PCR was designed with primers targeting fragments of 400 to 1,000 base pairs (bp) along the full length of the viral genome. The resulting reads were compared after sequencing with the Nanopore technology. Genome assembly revealed that the smaller fragments generated larger numbers of reads, allowing a more reliable coverage of the genome than those attained with primers targeting larger amplicons. Both new methodologies were efficient in amplification and sequencing.

Materials

Consumables:
Isopropanol
Beta-mercaptoethanol
Ethanol
Nuclease-free water
(primers from Table 1 and 2)
R9.4 Oxford MinION flowcell (FLO-MIN106)(Oxford Nanopore)
Commercial Kits:
    * Quick-DNA/RNA™ Viral MagBead (Zymo Research)
    * Q5® High-Fidelity 2X Master Mix (New England Biolabs)
    * AMPure XP beads (Beckman Coulter)
    * Ligation Sequencing kit SQK-LSK-109 (Oxford Nanopore)
    * Native Barcoding kits EXP-NBD104 and EXP-NBD114 (Oxford Nanopore)
    
Equipment:
Thermocycler
Magnetic stand (or automated extractor with magnetic block)
MinION Mk1B device (Oxford Nanopore)

Protocol materials

ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2140 / R2141Step 1
 
ReagentQ5 High-Fidelity PCR Kit - 200 rxnsNew England BiolabsCatalog #E0555LStep 11
 
Reagent Ampure XP beads Beckman CoulterCatalog #A63881Step 14
 
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854Step 19.1

Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)

This protocol uses    ReagentQuick-DNA/RNA Viral MagBeadZymo ResearchCatalog #R2140 / R2141  for extraction. 

All kit's solutions need to be prepared beforehand, as described by the kit. If you've already prepared the solution, go to step 2. This is a modified version of the kit's protocol. The original kit's protocol can be found here.

DNA/RNA Buffer:

Add Amount500 µL    of beta-mercaptoehtanol (user supplied) per 100 ml Viral DNA/RNA Buffer, (final 0.5% (v/v)

DNA/RNA Wash 1:

Kit R2140 --> Add Amount20 mL   of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

Kit R2141 --> Add  Amount80 mL   of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

DNA/RNA Wash 2:

Kit R2140 --> Add Amount30 mL   of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate

Kit R2141 --> Add Amount120 mL   of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate

DNA/RNA Shield™:

Kit R2140 --> Add Amount25 mL   of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate 

Kit R2141 --> Add Amount125 mL   of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate 

Proteinase K (20 mg/ml):

Kit R2140 --> Add Amount1.04 mL   of Proteinase K Storage Buffer to lyophilized Proteinase K (Amount20 mg  )

Kit R2141 --> Add Amount3.12 mL   of Proteinase K Storage Buffer to lyophilized Proteinase K (Amount60 mg  )

Mix by vortexing. Use immediately or store frozen aliquots.

Elute each dry SampleSample  into Amount400 µL   of 1X viral DNA/RNA buffer.

Our samples were dry rectal swabs of animals confirmed positive by a rapid test.
Samples were collected and stored in a centrifuge tube (without transporting media) at -20 ºC until processing.

For each sample, add   Amount10 µL   of beads and Amount4 µL   of proteinase K.

Obs: when working with many samples, one can always prepare a mixed solution with both components. Just make to keep the beads suspended while pipetting. 

Transfer the plate (or your tubes) to magnetic stand (user supplied). After the beads have pelleted, aspirate and discard the supernatant.

This protocol can be fast tracked by the use of an automated extractor. 
We use EXTRACTA 96 (Loccus). 

Perform one wash of the pellet (beads) with Amount250 µL   of MagBead DNA/RNA Wash 1 solution.

Perform one wash of the pellet (beads) with Amount250 µL   of MagBead DNA/RNA Wash 2 solution.

Add Amount250 µL   of 80% ethanol and mix well.
Transfer the sample (liquid and beads) to a new place/tube.

Pellet the beads and discard the the supernatant.

Add Amount50 µL   of nuclease-free water and mix well (this is the elution step).

Pellet the beads (magnetically) and transfer the supernatant (containing DNA/RNA) to a new plate/tube.

Multiplex PCR to obtain CPV genome fragments

This protocol uses 2 sets of oligos: one set amplifies ~400 bp sequences that overlap about 100 nucleotides with each other (Table 1);  the other amplifies ~1000 bp sequences that overlap about 100 nucleotides with each other (Table 2).  Primers are listed in the tables below and were design based on Canine parvovirus reference sequence NC_001539.1 (GenBank).

Primers should be mix into 2 pools for each set. To prepare a pool, add Amount5 µL   of each primer (at Concentration100 micromolar (µM)  ) as indicated. This will be your 10X (stock) solution. Working solution should be dilute to 1X concentration (Concentration10 micromolar (µM)  ).



NamePoolSequence (5’ – 3’)Size (nt)%GCTm
  CPV-400_0.7_LEFT*    1    ATGTCTGGCAACCAGTATACTG    22    45.50    60.30  
  CPV-400_1_LEFT    1    AACCAACTGACCAAGTTCACGT    22    45.45    60.80  
  CPV-400_1_RIGHT    1    GTTCCAGCGAACATCCTTTCCA    22    50.00    61.31  
  CPV-400_1.5_LEFT*    1    AGGTGGCGGGCTAATTGTG    19    57.90    59.50  
  CPV-400_2_LEFT    2    AAGAAACATGCAGAAAATGAAGCATT    26    30.77    59.73  
  CPV-400_2_RIGHT    2    CGTAGCCATTTACCAGTTGCTTG    23    47.83    60.67  
  CPV-400_3_LEFT    1    ATGGGGAAAAGATCAAGGCTGG    22    50.00    60.81  
  CPV-400_3_RIGHT    1    AGTGTGCTGACAATTTGTCTGTC    23    43.48    59.94  
  CPV-400_4_LEFT    2    GGGTGACTATATTAACATACAGACATAAGC    30    36.67    60.59  
  CPV-400_4_RIGHT    2    TCCTGGTTGTGCCATCATTTCA    22    45.45    60.68  
  CPV-400_5_LEFT    1    ACTTTGCGGGACTTGGTTAGTA    22    45.45    59.81  
  CPV-400_5_RIGHT    1    ACAACCAACATTACCCACAGCT    22    45.45    60.61  
  CPV-400_6_LEFT    2    CAGTTCTTTTTCATGGACCAGCA    23    43.48    59.93  
  CPV-400_6_RIGHT    2    AAACCAAAGTCTCCTGGAAGCT    22    45.45    60.01  
  CPV-400_7_LEFT    1    TGGATGTGAAGAAAGACCTGAACA    24    41.67    60.47  
  CPV-400_7_RIGHT    1    AACGCCAAGTTGGTTTGATTGT    22    40.91    60.01  
  CPV-400_8_LEFT    2    AGTGGACCTTGCACTGGAAC    20    55.00    60.20  
  CPV-400_8_RIGHT    2    GCTTCGTCGTGTTCTTTTGCAG    22    50.00    61.33  
  CPV-400_9_LEFT    1    AAATATCTTGGGCCTGGGAACA    22    45.45    59.87  
  CPV-400_9_RIGHT    1    ACTGCTCCATCACTCATTGGTG    22    50.00    60.80  
  CPV-400_10_LEFT    2    ACCACCTCATATTTTCATCAATCTTGC    27    37.04    60.91  
  CPV-400_10_RIGHT    2    TCAACCAATGACCAAGGTGTTACA    24    41.67    60.95  
  CPV-400_11_LEFT    1    GTGGTTGTAAATAATATGGATAAAACTGCA    30    30.00    60.00  
  CPV-400_11_RIGHT    1    TGTTCTATCCCATTGAAAATAATATCTCCA    30    30.00    59.80  
  CPV-400_12_LEFT    2    TGCCATTTACTCCAGCAGCTAT    22    45.45    60.01  
  CPV-400_12_RIGHT    2    TCCCATTTGAGTTACACCACGT    22    45.45    60.08  
  CPV-400_13_LEFT    1    TTTGCCTCAATCTGAAGGAGCT    22    45.45    60.14  
  CPV-400_13_RIGHT    1    ATCATTCGTTACAGGAAGGTTAAAGTT    27    33.33    59.83  
  CPV-400_14_LEFT    2    ACACCTGAGAGATTTACATATATAGCACA    29    34.48    60.63  
  CPV-400_14_RIGHT    2    ACCTTTCCACCAAAAATCTGAGTAAG    26    38.46    60.18  
  CPV-400_15_LEFT    1    CAAATGGTCAAATTTGGGATAAAGAATTTG    30    30.00    60.15  
  CPV-400_15_RIGHT    1    TTCTAGGTGCTAGTTGATATGTAATAAACA    30    30.00    59.56  
  CPV-400_16_LEFT    2    TGTTTATTACATATCAACTAGCACCTAGAA    30    30.00    59.56  
  CPV-400_16_RIGHT    2    TCTAAGGGCAAACCAACCAACC    22    50.00    61.20  
  CPV-400_17_LEFT    1    AGGTTTGTTAGATGGTATACAATAACTGT    29    31.03    59.61  
  CPV-400_17_RIGHT    1    AGCTTTAAATACTAATTTACCTTTCCACCA    30    30.00    60.50  
  CPV-400_17.5_RIGHT*    1    AAGTATCAATCTGTCTTTAAGGGG    24    37.50    60.10  
  CPV-400_18_LEFT*    2    TATAAGGTGAACTAACCTTACCATA    25    32.00    59.20  
  CPV-400_18_RIGHT*    2    TTAATATAATTTTCTAGGTGCTAGTTG    27    25.90    59.20  
Table 1. Primers targeting 400 bp amplicons 


 
NamePoolSequence (5'-3')Size (nt)%GCTm
  CPV-1000_1_LEFT   1CTGACCAAGTTCACGTACGTATGA  24    45.83    60.93  
  CPV-1000_1_RIGHT    1    TGTTCAGTGTAAAGTGTGCTGACA    24    41.67    61.18  
  CPV-1000_2_LEFT    2    GTGAATGGGTGACTATATTAACATACAGAC    30    36.67    60.73  
  CPV-1000_2_RIGHT    2    ACCAAACCAAAGTCTCCTGGAAG    23    47.83    60.95  
  CPV-1000_3_LEFT    1    AAGCAAATTGAACCAACTCCAGT    23    39.13    59.55  
  CPV-1000_3_RIGHT    1    GGTGGTGGTTTACTTCTTTTAGTTGG    26    42.31    60.90  
  CPV-1000_4_LEFT    2    CTAAGGACGCTAAAGATTGGGGG    23    52.17    61.00  
  CPV-1000_4_RIGHT    2    GTTCCTGTAGCAAATTCATCACCTG    25    44.00    60.77  
  CPV-1000_5_LEFT    1    CCATCTCATACTGGAACTAGTGGC    24    50.00    60.82  
  CPV-1000_5_RIGHT*    1    TGGATTCCAAGTATGAGAGGCTCT    24    45.83    61.21  
  CPV-1000_6_LEFT    2    AACCAAGACTTCATGTAAATGCACC    25    40.00    60.66  
  CPV-1000_6_RIGHT*    2    TGGATTCCAAGTATGAGAGGCTCT    24    45.83    61.21  
Table 2. Primers targeting 1000 bp amplicons 

We used ReagentQ5 High-Fidelity PCR Kit - 200 rxnsNew England BiolabsCatalog #E0555L   to perform PCR. 

Reactions should be set up independent for each pool, with a final volume of Amount25 µL  , as bellow:

Reactions for 400 bp products:                                      
        * Master Mix 2X         - Amount12.5 µL  
        * Primer pool 1 or 2   -   Amount1.6 µL    
        * Nuclease-free H20 -  Amount5.9 µL  
        * DNA                         -   Amount5.0 µL  

Reactions for 1000 bp products:                                      
        * Master Mix 2X         - Amount12.5 µL  
        * Primer pool 1 or 2   -  Amount0.4 µL    
        * Nuclease-free H20 -  Amount7.1 µL  
        * DNA                         -  Amount5.0 µL  

The final concentration in a reaction should be Concentration15 nanomolar (nM)   per primer. Therefore, the volume of primers used in a reaction will vary according with the number of primers in the pool. More about this subject can be found in Quick et al. (2017).

PCR's run method should be set as:
        * Temperature98 °C   for Duration00:03:00  
        * 35 cycles of (Temperature98 °C   for Duration00:00:15  ; Temperature63 °C   for Duration00:05:00  )
        * Hold at Temperature4 °C  

8m 15s

Purifying PCR Products with AMPure XP beads (Beckman Coulter)

Mix amplification products of Pool 1 and Pool 2 (final volume Amount50 µL  ).

We purify the fragments with Reagent Ampure XP beads Beckman CoulterCatalog #A63881  , using a modified protocol (original protocol ca be found here).

This is step can also be fast-tracked by the use of an automated system.

Add an equal volume of AMPure XP per sample (Amount50 µL  ).

After biding, on a magnetic stand, wash beads once with Concentration80 % (v/v)   ethanol.

Elute DNA with Amount20 µL   of (sequencing appropriate) buffer.

Transfer DNA to a new plate/tube.

Quantify your DNA.

We used ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854  
Equipment
Qubit™ 3 Fluorometer
NAME
Fluorometer for nucleic acid quantitation
TYPE
Invitrogen
BRAND
Q33216
SKU
https://www.thermofisher.com/order/catalog/product/br/en/Q33216
LINK
Fluorometer for nucleic acid quantitation
SPECIFICATIONS
 

Library preparation & sequencing

To prepare a library, use the Ligation and Barcoding kits (following manufacturer's instructions).

We used Ligation Sequencing kit SQK-LSK-109 and Native Barcoding kits EXP-NBD104 and EXP-NBD114 (Oxford Nanopore)

Load library on your flow cell and sequence your sample.

We used R9.4 Oxford MinION flow cell (FLO-MIN106) and MinION Mk1B device for sequencing.

Protocol references

Quick, J., Grubaugh, N. D., Pullan, S. T., Claro, I. M., Smith, A. D., Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T. F., Beutler, N. A., Burton, D. R., Lewis-Ximenez, L. L., de Jesus, J. G., Giovanetti, M., Hill, S. C., Black, A., Bedford, T., Carroll, M. W., Nunes, M., Alcantara, L. C., Jr, … Loman, N. J. (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature protocols, 12(6), 1261–1276. https://doi.org/10.1038/nprot.2017.066

Name	Pool	Sequence (5’ – 3’)	Size (nt)	%GC	Tm
CPV-400_0.7_LEFT*	1	ATGTCTGGCAACCAGTATACTG	22	45.50	60.30
CPV-400_1_LEFT	1	AACCAACTGACCAAGTTCACGT	22	45.45	60.80
CPV-400_1_RIGHT	1	GTTCCAGCGAACATCCTTTCCA	22	50.00	61.31
CPV-400_1.5_LEFT*	1	AGGTGGCGGGCTAATTGTG	19	57.90	59.50
CPV-400_2_LEFT	2	AAGAAACATGCAGAAAATGAAGCATT	26	30.77	59.73
CPV-400_2_RIGHT	2	CGTAGCCATTTACCAGTTGCTTG	23	47.83	60.67
CPV-400_3_LEFT	1	ATGGGGAAAAGATCAAGGCTGG	22	50.00	60.81
CPV-400_3_RIGHT	1	AGTGTGCTGACAATTTGTCTGTC	23	43.48	59.94
CPV-400_4_LEFT	2	GGGTGACTATATTAACATACAGACATAAGC	30	36.67	60.59
CPV-400_4_RIGHT	2	TCCTGGTTGTGCCATCATTTCA	22	45.45	60.68
CPV-400_5_LEFT	1	ACTTTGCGGGACTTGGTTAGTA	22	45.45	59.81
CPV-400_5_RIGHT	1	ACAACCAACATTACCCACAGCT	22	45.45	60.61
CPV-400_6_LEFT	2	CAGTTCTTTTTCATGGACCAGCA	23	43.48	59.93
CPV-400_6_RIGHT	2	AAACCAAAGTCTCCTGGAAGCT	22	45.45	60.01
CPV-400_7_LEFT	1	TGGATGTGAAGAAAGACCTGAACA	24	41.67	60.47
CPV-400_7_RIGHT	1	AACGCCAAGTTGGTTTGATTGT	22	40.91	60.01
CPV-400_8_LEFT	2	AGTGGACCTTGCACTGGAAC	20	55.00	60.20
CPV-400_8_RIGHT	2	GCTTCGTCGTGTTCTTTTGCAG	22	50.00	61.33
CPV-400_9_LEFT	1	AAATATCTTGGGCCTGGGAACA	22	45.45	59.87
CPV-400_9_RIGHT	1	ACTGCTCCATCACTCATTGGTG	22	50.00	60.80
CPV-400_10_LEFT	2	ACCACCTCATATTTTCATCAATCTTGC	27	37.04	60.91
CPV-400_10_RIGHT	2	TCAACCAATGACCAAGGTGTTACA	24	41.67	60.95
CPV-400_11_LEFT	1	GTGGTTGTAAATAATATGGATAAAACTGCA	30	30.00	60.00
CPV-400_11_RIGHT	1	TGTTCTATCCCATTGAAAATAATATCTCCA	30	30.00	59.80
CPV-400_12_LEFT	2	TGCCATTTACTCCAGCAGCTAT	22	45.45	60.01
CPV-400_12_RIGHT	2	TCCCATTTGAGTTACACCACGT	22	45.45	60.08
CPV-400_13_LEFT	1	TTTGCCTCAATCTGAAGGAGCT	22	45.45	60.14
CPV-400_13_RIGHT	1	ATCATTCGTTACAGGAAGGTTAAAGTT	27	33.33	59.83
CPV-400_14_LEFT	2	ACACCTGAGAGATTTACATATATAGCACA	29	34.48	60.63
CPV-400_14_RIGHT	2	ACCTTTCCACCAAAAATCTGAGTAAG	26	38.46	60.18
CPV-400_15_LEFT	1	CAAATGGTCAAATTTGGGATAAAGAATTTG	30	30.00	60.15
CPV-400_15_RIGHT	1	TTCTAGGTGCTAGTTGATATGTAATAAACA	30	30.00	59.56
CPV-400_16_LEFT	2	TGTTTATTACATATCAACTAGCACCTAGAA	30	30.00	59.56
CPV-400_16_RIGHT	2	TCTAAGGGCAAACCAACCAACC	22	50.00	61.20
CPV-400_17_LEFT	1	AGGTTTGTTAGATGGTATACAATAACTGT	29	31.03	59.61
CPV-400_17_RIGHT	1	AGCTTTAAATACTAATTTACCTTTCCACCA	30	30.00	60.50
CPV-400_17.5_RIGHT*	1	AAGTATCAATCTGTCTTTAAGGGG	24	37.50	60.10
CPV-400_18_LEFT*	2	TATAAGGTGAACTAACCTTACCATA	25	32.00	59.20
CPV-400_18_RIGHT*	2	TTAATATAATTTTCTAGGTGCTAGTTG	27	25.90	59.20

Name	Pool	Sequence (5'-3')	Size (nt)	%GC	Tm
CPV-1000_1_LEFT	1	CTGACCAAGTTCACGTACGTATGA	24	45.83	60.93
CPV-1000_1_RIGHT	1	TGTTCAGTGTAAAGTGTGCTGACA	24	41.67	61.18
CPV-1000_2_LEFT	2	GTGAATGGGTGACTATATTAACATACAGAC	30	36.67	60.73
CPV-1000_2_RIGHT	2	ACCAAACCAAAGTCTCCTGGAAG	23	47.83	60.95
CPV-1000_3_LEFT	1	AAGCAAATTGAACCAACTCCAGT	23	39.13	59.55
CPV-1000_3_RIGHT	1	GGTGGTGGTTTACTTCTTTTAGTTGG	26	42.31	60.90
CPV-1000_4_LEFT	2	CTAAGGACGCTAAAGATTGGGGG	23	52.17	61.00
CPV-1000_4_RIGHT	2	GTTCCTGTAGCAAATTCATCACCTG	25	44.00	60.77
CPV-1000_5_LEFT	1	CCATCTCATACTGGAACTAGTGGC	24	50.00	60.82
CPV-1000_5_RIGHT*	1	TGGATTCCAAGTATGAGAGGCTCT	24	45.83	61.21
CPV-1000_6_LEFT	2	AACCAAGACTTCATGTAAATGCACC	25	40.00	60.66
CPV-1000_6_RIGHT*	2	TGGATTCCAAGTATGAGAGGCTCT	24	45.83	61.21

Public workspaceSequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1

Sequencing of Canine Parvovirus (CPV) from Rectal Swab Samples V.1