Overview of Hepatitis C virus assays
Transcript of Overview of Hepatitis C virus assays
COBAS® AmpliPrep/COBAS® TaqMan® HCV Test
Overview molecular HCV analyses Viral load and genotyping
Versant HCV Genotype 2.0 Assay (INNO-LiPA)
Overview molecular HCV analyses sequence analysis
RNA isolation RT-PCR Plasma
Population sequencing
Cloning
Ultradeep/next gen seq
Phenotyping Amplimers
Sensitivity for detection mutations: -Population sequencing: ~10-25% -Clonal analysis: ~5% -Ultra Deep Sequencing: ~1%
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HCV genotyping for confirmation of standard assays, e.g., HCV-LiPA
NS5B small (~325 bp)
BLAST Search: homology score
Phylogenetic analysis
HCV (sub)type
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HCV Genotyping: Phylogenetic tree 1a._.6001.EF407422 (0.0205)
1a.US.BID-V105.EU155215 (0.0151)
1a.DE.BID-V35.EU155380 (0.0160)
1a._.7065.EF407455 (0.0248)
P176M0344. (0.0206)
1a.CH.BID-V227.EU482853 (0.0240)
P176M0251B2. (0.0125)
P176M0358B2. (0.0143)
1c.IN.AY051292.AY051292 (0.0242)
1c.IN.Khaja1.AY651061 (0.0262)
1b._.MD27.AF207768 (0.0247)
1b.RU.N589.AY587844 (0.0227)
1b.US.BID-V369.EU155257 (0.0217)
1b.US.BID-V352.EU155306 (0.0296)
1b.JP.MD1-911.AF165046 (0.0303)
1b._.MD32.AF207773 (0.0323)
1b._.MD29.AF207770 (0.0285)
1b.JP.HCV-KT1.AB426117 (0.0303)
1b.JP.MD8-1114.AF165060 (0.0202)
1b.US.BID-V370.EU482839 (0.0397)
5a.GB.EUH1480.Y13184 (0.0369)
5a.ZA.SA13.AF064490 (0.0225)
2a._.G2AK1.AF169003 (0.0297)
2a._.MD2A-2.AF238482 (0.0234)
2a._.MD2A-4.AF238483 (0.0270)
2a._.MD2A-1.AF238481 (0.0320)
2a.JP.JCH-6.AB047645 (0.0244)
2c._.BEBE1.D50409 (0.0688)
2k.MD.VAT96.AB031663 (0.0766)
2b._.JPUT971017.AB030907 (0.0415)
2b._.MD2B-1.AF238486 (0.0316)
2b.JP.HC-J8.D10988 (0.0278)
3a._.CB.AF046866 (0.0378)
3a._.NZL1.NC_009824 (0.0204)
3a.DE.HCVCENS1.X76918 (0.0248)
P176M0359B2. (0.0198)
3a._.K3A.D28917 (0.0430)
3b.IN.RG245.AY515261 (0.0451)
3b.JP.HCV-Tr.D49374 (0.0499)
3k.ID.JK049.D63821 (0.0448)
P176M0063A2. (0.0084)
P176M0293. (0.0035)
P176M0305. (0.0658)
4a._.F7157.DQ418788 (0.0142)
4a._.L835.DQ418789 (0.0155)
4a._.F753.DQ418787 (0.0283)
4a.EG.ED43.Y11604 (0.0416)
4d._.03-18.DQ418786 (0.0734)
7a.CA.QC69.EF108306 (0.1749)
6h.VN.VN004.D84265 (0.0891)
6j.TH.C-0667.DQ835761 (0.0207)
6j.TH.Th553.DQ835769 (0.0209)
6i.TH.C-0159.DQ835762 (0.0260)
6i.TH.Th602.DQ835770 (0.0274)
6m.TH.B4/ 92.DQ835767 (0.0216)
6m.TH.C-0208.DQ835763 (0.0170)
6m.TH.C-0185.DQ835765 (0.0132)
6m.TH.C-0192.DQ835766 (0.0105)
P176M0354. (0.0497)
P176M0363. (0.0565)
6n.CN.KM42.DQ278894 (0.0231)
6n.TH.D86/ 93.DQ835768 (0.0244)
6k.CN.KM41.DQ278893 (0.0311)
6k.CN.KM45.DQ278891 (0.0342)
6k.VN.VN405.D84264 (0.0775)
6l.US.537796.EF424628 (0.0350)
P176M0331A2. (0.0252)
P176M0332A2. (0.0185)
P176M0339. (0.0143)
6b._.Th580.NC_009827 (0.1139)
P176M0337. (0.0213)
P176M0353. (0.0204)
P176M0346. (0.0278)
P176M0345. (0.0347)
P176M0352. (0.0321)
P176M0342. (0.0367)
P176M0343. (0.0512)
6.CN.GZ52557.DQ278892 (0.1203)
6g.HK.HK6554.DQ314806 (0.0290)
6g.ID.JK046.D63822 (0.0273)
6e.CN.GX004.DQ314805 (0.0346)
P176M0340. (0.0240)
P176M0347. (0.0295)
P176M0334A2. (0.0330)
P176M0338. (0.0403)
P176M0355A3. (0.0403)
P176M0336. (0.1361)
6c.TH.Th846.EF424629 (0.0878)
6d.VN.VN235.D84263 (0.0873)
6f.TH.C-0044.DQ835760 (0.0061)
6f.TH.C-0046.DQ835764 (0.0058)
P176M0351. (0.0861)
6q.CA.QC99.EF424625 (0.0272)
P176M0350. (0.0204)
P176M0349. (0.0273)
6p.CA.QC216.EF424626 (0.0365)
P176M0341. (0.0439)
P176M0333A2. (0.0638)
6o.CA.QC227.EF424627 (0.0951)
6t.VN.TV241.EF632069 (0.0295)
6t.VN.TV249.EF632070 (0.0344)
6t.VN.VT21.EF632071 (0.0249)
P176M0348. (0.0876)
1a
1c 1b
5a 2a 2c
2b 3a
3b 3k 4a 4d 6a-t
Clinical samples (boxed) are compared to a large reference set of all HCV genotypes
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Antiviral treatments
NS3 protease/helicase NS5B RNA polymerase NS5A
5’RACE
Availability of PCR assays or Sanger and/or NGS
Target GT1 GT2 GT3 GT4 GT5 GT6
5’UTR-core + + + + + - NS3-4a + + + + + + NS5a + + + + + + NS5B + + + + + + 5’UTR RACE + - - + - -
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HCV Amplification & Sequencing
• No prior subtyping required, only HCV type (except for GT6)
minimal amplification bias
• Using high-fidelity enzymes (Roche Transcriptor & Expand)
minimal polymerase errors
• Detection limit: 1,000-10,000 IU/ml
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Deep sequencing
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Illumina MiSeq
Data analysis and reporting pipeline
Lab report (extensive & complex)
Clinical site report (interpretation)
Total RNA sequencing HCV
First strand cDNA synthesis Second strand cDNA synthesis SPIA amplifciation
Ovation® RNA-Seq System v2 Library preparation
Data analysis: sequence annotation
Total RNA
Illumina sequencing
RNA isolation
Plasma
Extract HCV-matching reads
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Summary DDL HCV services
• Viral load testing • Genotyping HCV
– Siemens Versant HCV Genotyping LiPA 2.0 – NS5B (samples failing standard typing methods)
• Sequencing – GT1 to 6, NS3-4A, NS5A, NS5B covered (type-specific) – Population – Deep sequencing (Illumina MiSeq)
• Target-specific amplicons • Total RNA/full genome
– Clonal
• HCV replicon-based phenotyping • Data management and tailor-made reporting
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