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

[DESCRIPTION]
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.



[STEPS]
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
2. Elute each dry   into 400 µL of 1X viral DNA/RNA buffer.

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
3. For each sample, add   10 µL of beads and 4 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. 

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
4. Perform one wash of the pellet (beads) with 250 µL of MagBead DNA/RNA Wash 1 solution.

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
5. Perform one wash of the pellet (beads) with 250 µL of MagBead DNA/RNA Wash 2 solution.

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
6. Add 250 µL of 80% ethanol and mix well.
Transfer the sample (liquid and beads) to a new place/tube.

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
8. Add 50 µL of nuclease-free water and mix well (this is the elution step).
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
3.1. 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). 


SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
7. Pellet the beads and discard the the supernatant.
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1.5. Proteinase K (20 mg/ml):

Kit R2140 --> Add 1.04 mL of Proteinase K Storage Buffer to lyophilized Proteinase K (20 mg)

Kit R2141 --> Add 3.12 mL of Proteinase K Storage Buffer to lyophilized Proteinase K (60 mg)

Mix by vortexing. Use immediately or store frozen aliquots.
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1.4. DNA/RNA Shield™:

Kit R2140 --> Add 25 mL of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate 

Kit R2141 --> Add 125 mL of nuclease-free water (user supplied) to 2X DNA/RNA Shield™ concentrate 

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1.3. DNA/RNA Wash 2:

Kit R2140 --> Add 30 mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate

Kit R2141 --> Add 120 mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 2 concentrate
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1.2. DNA/RNA Wash 1:

Kit R2140 --> Add 20 mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

Kit R2141 --> Add  80 mL of isopropanol (2-propanol PA, user supplied) to MagBead DNA/RNA Wash 1 concentrate

SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1.1. DNA/RNA Buffer:

Add 500 µL  of beta-mercaptoehtanol (user supplied) per 100 ml Viral DNA/RNA Buffer, (final 0.5% (v/v)
   
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
1. This protocol uses      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.
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
9. Pellet the beads (magnetically) and transfer the supernatant (containing DNA/RNA) to a new plate/tube.
SECTION: Multiplex PCR to obtain CPV genome fragments
10. 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 5 µL of each primer (at 100 micromolar (µM)) as indicated. This will be your 10X (stock) solution. Working solution should be dilute to 1X concentration (10 micromolar (µM)).



     Table 1. Pool 1 (Primers targeting


 
     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   


SECTION: Multiplex PCR to obtain CPV genome fragments
11. We used    to perform PCR. 

Reactions should be set up independent for each pool, with a final volume of 25 µL, as bellow:

Reactions for 400 bp products:                                      
        * Master Mix 2X         - 12.5 µL
        * Primer pool 1 or 2   -   1.6 µL  
        * Nuclease-free H20 -  5.9 µL
        * DNA                         -   5.0 µL

Reactions for 1000 bp products:                                      
        * Master Mix 2X         - 12.5 µL
        * Primer pool 1 or 2   -  0.4 µL  
        * Nuclease-free H20 -  7.1 µL
        * DNA                         -  5.0 µL

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
14. We purify the fragments with   , using a modified protocol (original protocol ca be found here).

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
19. Quantify your DNA.
SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
19.1. We used   
 
 


SECTION: Library preparation &amp; sequencing
20. 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)

SECTION: Library preparation &amp; sequencing
21. Load library on your flow cell and sequence your sample.



SECTION: Library preparation &amp; sequencing
21.1. We used R9.4 Oxford MinION flow cell (FLO-MIN106) and MinION Mk1B device for sequencing.







SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
13. Mix amplification products of Pool 1 and Pool 2 (final volume 50 µL).

SECTION: Multiplex PCR to obtain CPV genome fragments
12. PCR's run method should be set as:
        * 98 °C for 3 min
        * 35 cycles of (98 °C for 15 s; 63 °C for 5 min)
        * Hold at 4 °C

SECTION: Multiplex PCR to obtain CPV genome fragments
11.1. The final concentration in a reaction should be 15 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).

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
14.1. This is step can also be fast-tracked by the use of an automated system.
20. 
SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
15. Add an equal volume of AMPure XP per sample (50 µL).

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
16. After biding, on a magnetic stand, wash beads once with 80 % (v/v) ethanol.

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
17. Elute DNA with 20 µL of (sequencing appropriate) buffer.

SECTION: Purifying PCR Products with AMPure XP beads (Beckman Coulter)
18. Transfer DNA to a new plate/tube.
SECTION: Nucleic Acid Extraction using Quick-DNA/RNA Viral MagBead (Zymo Research)
2.1. 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.