New technologies reaching the clinic
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1 New technologies reaching the clinic Martin Däumer May 31, 2018
2 Deep-sequencing Standard Sanger-sequencing...PQIYMDDHTRE... Ultra-deep-sequencing...PQIYMDDHTRE......PQIYMDDHTRE......PQIYVDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE...
3 Developments in high-throughput sequencing
4 The players
5 Ion Torrent PGM (Personal Genome Machine) The chip is the machine
6 Ion Torrent workflow
7 Specifications
8 Vela Next Generation Sequencing NGS automation for the IVD routine laboratory Sentosa SQ HCV/HIV Genotyping Assay Sentosa Link Sample ID Download Samples 4 Validated Sample Materials; 2 Extraction Kits Sentosa SX101 Multi-purpose: Lysis, Extraction, Library Prep; Open-channel Sentosa ST401 Template Prep Sentosa SQ301 IVD Sequencing, Open-channel Sentosa SQ Reporter Data QC Automated Reporting Sentosa Link Result Upload Key Features CE-IVD Ready to use in 2 weeks from installation IT Connectivity Sample Tracking Control System Automation Ready to use Reagents Actionable Design Data Analysis Data Reporting
9 Sentosa SQ Virology Panels HCV and HIV Panel Specifications Parameters HCV HIV Regualtory Status CE-IVD CE-IVD Target Genes NS3, NS5A, NS5B Protease, Reverse Transcriptase, Integrase Genotyping capabilities Median Coverage/ Sample 1a, 1b, 2, 3, 4, 5, 6 (Analysis of all clinically relevant RAV s included) 200x for genotyping 500x for variants calling HIV-1 group M (subtypes AG, B, C, D, AE, F, G, CRF02, CRF03, A and recombinant A) 1,000x for genotyping and variant calling Sample Input Plasma (EDTA / CPD / ACD) and Serum Plasma (EDTA / CPD / ACD) Sample Throughput 15 patient samples and 1 system control 15 patient samples and 1 system control Analytical Sensitivity (Limit of detection) Analytical Specificity 1,000 HCV IU/mL for Genotypes 1a, 1b, 2, 3 and 4 2,000 HCV IU/mL for Genotypes 5, 6 No cross-reaction with Dengue virus, HAV, HBV, HIV, CMV, EBV, BKV, human genomic DNA 1,000 copies/ml No cross reactivity with Chlamydia trachomatis, Staphylococcus aureus, Mycobacterium smegmatis, HCV, Neisseria meningitidis, HHV6, BKV, EBV, Hep A, HBV CMV VZV HSV1 and HSV2
10 Sentosa SQ Workflow 15 clinical samples per run, 4 runs (60 samples) per week Monday Tuesday Wednesday Thursday Friday AM PM AM PM AM PM AM PM AM PM 1 SX101 ST401 SQ301 2 SX101 ST401 SQ301 3 SX101 ST401 SQ301 4 SX101 ST401 SQ301 Costs per sample (when running 15 samples): 110,-
11 Illumina platforms
12 MiSeq Personal Sequencing System Cluster generation and Sequencing-by-Synthesis
13 Library preparation using Nextera TM XT tagmentation Easy library preparation Fast - less than 15 minutes hands-on time Only 1ng DNA per sample needed 384 indices available Normalization step included
14 Library Normalization
15 Third generation sequencing
![fragment size (longest reported read: 892 kb [1D])](/docs-images/84/89563386/images/16-2.jpg "Starter kit: $1.")
16 MinION Nanopore technology output: up to 21Gb (1D at 400 bps in 48 hours) read length: dependent on fragment size (longest reported read: 892 kb [1D]) Starter kit: $1.000 (1X minion, reagents for two runs)
17 Oxford Nanopore s technology
18 MinION Nanopore technology HIV-1 pol amplicon, 1.35kb
19 Oxford Nanopore s error rate
20 Rolling Circle Amplification random priming isothermal amplification by phi29 polymerase
21 RCA Approach: Final product copy 1 copy 2 copy n kb
22 RCA Approach: Analysis copy 1 copy 2 copy n kb Individual copies are detected in the sequenced reads and aligned against each other copy 1 x x copy 2 x x x... copy n x x x A consensus sequence is generated consensus
23 Results Basecalling errors at different sensitivity cut-offs:
24 Outlook: Direct RNA sequencing with ONT
25 Drug resistance testing (HIV) using Illumina s MiSeq
26 Experimental setup HIV genome PRRT IN ENV whole genome
27 Fragmentation
28 Automated Nextera XT library preparation
29 putting things together mapping PRRT IN ENV Reference sequences
30 Coverage ~10K to 20K ~10K to 20K PR/RT ENV ~7500 full V3 loops
31 Validation isolation NGS Sanger pooling rt-pcr/ nested pcr fragmentation & indexing sequencing 400 samples (PR/RT) sequencing analysis analysis comparison
32 PR/RT # resistance mutations found
33 Viral load cop/ml How much virus is relevant? 1% 1%
34 Number of mutations not detected by the respective sequencing approach (Sanger vs NGS and vice versa) at 2% and 10% NGS minority cutoff 0 0 n=30
35 Sanger vs NGS (same amplicon)
36 Workflow NGS RNA/DNA Total NA extraction Sanger Library preparation: fragmentation & indexing Sequencing reaction analysis PCR errors (Sequencing Errors) rt-pcr/ nested pcr RNA vs. DNA: viable vs non-viable RT/PCR errors, recombination Potential error sources editing Taq-cycle reaction, sequencing analysis
37 Deviations from the main variant (amino acid; in %) HIV-1 protease (clone) RT-PCR/nested PCR 1% H: SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity /Platinum Taq High Fidelity (Invitrogen/Thermo) QH: QIAGEN OneStep Ahead RT-PCR Kit/Platinum Taq High Fidelity QQ: QIAGEN OneStep Ahead RT-PCR Kit/QIAGEN HotStarTaq polymerase Q: QIAGEN OneStep RT-PCR Kit/QIAGEN HotStarTaq polymerase
38 Case report: T., T.-L. *1974 TDF/FTC/RPV Resistance tests, retrospective
39 Case report: T., T.-L. *1974 Date Mutation (%) TDF FTC RPV 05/2014 E138K (100%) 07/2014 E138K (100%) Y115F (5,6%) M184V (100%) V108I (100%) K219R (0,4%) 10/2014 K70E (1,8%) E138K (100%) Y115F (100%) V179L (1,6%) M184V (100%) V108I (100%) K219R (2,8%) 02/2015 K70E (100%) E138R (100%) Y115F (100%) V179L (100%) M184V (100%) V108I (100%) K219R (82%)
40 The deeptypehiv report generator Muts >10% Muts >2%<10% Patient identification and therapy data Scored mutations per drug Information on coverage Detailed information mut and wt frequency Data modules
41 The deeptypehiv report generator Use tick boxes for visualization of resistance situation: Susceptible Limited susceptibility Intermediate Resistant
42 The deeptypehiv report generator Preconfigured report components available
43 The deeptypehiv report generator, integrated HIV-GRADE interpretation
44 The deeptypehiv report
45 The deeptypehiv report
46 Cost per sample calculation Illumina MiSeq (2x250) (reagents only) 25 samples per week PRRT plus Integrase plus env V3 : nts Coverage: X sum: 1,7 Gb, spare capacity: 6-7 Gb (2x250nts) Sample and library preparation: 39,-/sample* Sequencing: 53,-/sample* total 92,-/sample (incl. of VAT) Bioinformatician not included ( There are some things money can t buy... for the rest there s Mastercard ) *based on Illumina s list prices in Germany 2017
47 Cost per raw Megabase
48 Acknowledgments Bettina Welter Kirsten Becker Anna Memmer Nina Engel Anja Laux Alexander Thielen Bernhard Thiele
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