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Evaluation of direct metagenomics and target enriched approaches for high-throughput sequencing of field rabies viruses

Anna Orłowska
Anna Orłowska
, 
Ewelina Iwan
Ewelina Iwan
, 
Marcin Smreczak
Marcin Smreczak
 and 
Jerzy Rola
Jerzy Rola
   | Nov 16, 2019
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Published Online: Nov 16, 2019
Page range: 471 - 479
Received: Mar 06, 2019
Accepted: Nov 04, 2019
DOI: https://doi.org/10.2478/jvetres-2019-0067
Keywords
© 2019 A. Orłowska et al. published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

A schematic of the RABV genome indicating position of five ORFs and primers
A schematic of the RABV genome indicating position of five ORFs and primers

Fig. 2

Gel analysis of representative purified products of amplification. A – first round (amplicons A, B, and C). B – second round (amplicons A1, A2, B1, B2, C1, and C2)
Gel analysis of representative purified products of amplification. A – first round (amplicons A, B, and C). B – second round (amplicons A1, A2, B1, B2, C1, and C2)

Primers employed for RT-PCR of field RABV samples

Amplicon Primer name Primer sequence 5′-3′ Location genome in RABV Amplicon size
A RVA_forward ATGGATGCCGACAAGATTGTATT 1–23 4499
RVA_reverse CAGGGGGTGCATCAGGGGAAT 4478–4499
B RVB_forward ATCCCAGAGATGCAATCATCC 4418–4439 3860
RVB_reverse TGAGTAGAATGGTAGGACTGGCACC 8251–8276
C RVC_forward GAACCCAGATCTTGGAGAGAGAA 8172–8195 3631
RVC_reverse TTCGGATTCAAGATCTTGTTTT 11779–11801
A1 RVA_forward as above 2267
RVA1_reverse TGGAATTTCTTGGAATTGGCCAAAGC 2241–2267
A2 RVA2_forward GCTCATGACGGATCCAAACTCCC 2193–2216 2300
RVA_reverse as above
B1 RVB_forward as above 2330
RVB1_reverse GATTCAGGAATCTCAAAGATTTGCGT 6724–6750
B2 RVB2_forward TTGACTCCTTATATCAAAACCCAGA 6640–6665 1636
RVB_reverse as above
C1 RVC_forward as above 2016
RVC1_reverse GTCATGGTTCTAGCTGCATGGCG 10155–10188
C2 RVC2_forward ATGAGGCAGGTGCTGGGTG 10054–10073 1750
RVC_reverse as above

Details of samples in the comparative study of extraction methods. RNA concentration, virus detection using real-time RT-PCR, and the number of reads obtained during whole-genome sequencing

Group Isolate Sample origin Collection date Extraction method /RT-PCR procedure Concentration of dsDNA after clean-up (Qubit HS) (ng/µL) Verification of HTS library Total number of reads Number of viral reads % of viral reads Number of RABV reads (centrifuge) Number of contigs Average coverage
Direct metagenomic approach I 767121097L red fox 1997 A – QIAmp Viral RNA Mini 1.45 + 22,250 117 0.0525 3 - -
965180404L red fox 2004 Kit/ RT + amplification of dsDNA with Klenow 1.06 + 15,282 67 0.438 0 - -
1045120899L red fox 1999 fragment 1.08 + 158,723 496 0.31 8 - -
II 767121097L red fox 1997 5.06 − - - - - -
965180404L red fox 2004 B – Direct-zol RNA MiniPrep 1.21 + 160,354 1,1170 6.965 132 19 3.5
1045120899L red fox 1999 Zymo amplification Research/ of RT dsDNA + with 1.15 + 200,756 4,218 2.101 133 6 2.5
1321180108L red fox 2008 Klenow fragment 1.2 + 517,696 70,436 13.605 1,359 1 32
1379120910L red fox 2010 2.21 + 948,295 68,491 7.222 4,765 3 152
III 1996181013L* red fox 2013 1.18 + 839,440 47,118 5.613 28,116 1 495
1992121113L red fox 2013 13.4 + 423,115 1,417 0.334 62 9 2
1739120912L red fox 2012 2.93 + 4,216,387 14,103 0.334 657 1 15
1679180512L* red fox 2012 C – TRIzol/ chloroform/ethanol/ 2.04 + 4,543,264 21,683 0.4772 2,935 1 71
1577121111L red fox 2011 RT+ amplification of dsDNA 4.28 + 1,058,492 5,313 0.05019 564 1 13
1525180711L red fox 2011 with Klenow fragment 4.38 + 849,062 2,023 0.238 23 3 1.5
1391180910L red fox 2010 3.86 + 2,401,009 8,487 0.3534 10 3 1
EBLV-1 Eptesicus serotinus 2018 + 460,751 178,828 38.812 32,232 1 571
RABV-enriched approach IV 1045120899L as above as above 16.8 + 2,697,616 2,560,850 94.930 1,342,569 1 38,039
965180404L 56.5 + 2,951,939 2,765,165 93.67 1,503,424 1 41,961
767121097L 76.6 + 3,106,953 2,871,569 92.42 1,686,411 1 41,697
1379120910L 55.2 + 754,635 711,112 94.23 372,141 1 11,346
1321180108L 25.1 + 1,115,667 951,915 85.322 557,949 3 5,962
1525180711L 75.5 + 869,224 824,305 94.832 463,772 1 12,144
1739120912L 48.5 + 682,577 645,311 94.54 362,514 1 9,500
1996181013L* 65.4 + 760,390 718,626 94.507 423,322 1 10,144
1577121111L 81.4 + 904,532 846,126 93.54 507,207 1 12,122
1992121113L 75.9 + 978,751 922,826 94.286 525,370 1 13,536
1391180910L 47.3 + 985,144 943,962 95.819 539,481 1 13,075
2191180915L red fox 2015 C – TRIzol/chloroform/ 93 + 967,059 908,214 93.915 528,055 1 13,089
1990121113L red fox 2013 ethanol + virus enrichment 70.2 + 893,272 834,573 93.428 464,788 1 12,217
2176120515L red fox 2015 87.6 + 1,294,593 1,196,143 92.395 737,332 1 17,535
2068180814L red fox 2014 69.6 + 1,192,314 1,112,910 93.340 65,4514 1 16,190
2214181115L red fox 2015 87.6 + 1,230,082 1,140,045 92.680 709,820 1 16,316
2067120814L red fox 2014 62 + 1,265,627 1,150,880 90.933 703,662 1 17,091
2066120814L red fox 2014 68.4 + 1,934,161 1,741,947 90.062 1,062,064 1 25,144
2226120916L red fox 2016 84 + 1,133,386 1,011,316 89.229 633,117 1 14,546
2235181116L red fox 2016 103 + 1,947,768 1,828,286 93.865 1,119,736 1 26,791
2236181216P dog 2016 81.2 + 612,073 564,607 92.245 346,010 1 7,980
2237120117L red fox 2017 96.4 + 1,336,571 1,225,265 91.672 758,770 1 16,869
2238181117K cat 2017 62 + 1,034,833 918,601 88.768 562,828 1 12,576
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