New technologies reaching the clinic Martin Däumer May 31, 2018
Deep-sequencing Standard Sanger-sequencing...PQIYMDDHTRE... Ultra-deep-sequencing...PQIYMDDHTRE......PQIYMDDHTRE......PQIYVDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE......PQIYMDDHTRE...
Developments in high-throughput sequencing
The players
Ion Torrent PGM (Personal Genome Machine) The chip is the machine
Ion Torrent workflow
Specifications
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
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
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,-
Illumina platforms
MiSeq Personal Sequencing System Cluster generation and Sequencing-by-Synthesis
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
Library Normalization
Third generation sequencing
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)
Oxford Nanopore s technology
MinION Nanopore technology HIV-1 pol amplicon, 1.35kb
Oxford Nanopore s error rate
Rolling Circle Amplification random priming isothermal amplification by phi29 polymerase
RCA Approach: Final product copy 1 copy 2 copy n 80-100 kb
RCA Approach: Analysis copy 1 copy 2 copy n 80-100 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
Results Basecalling errors at different sensitivity cut-offs:
Outlook: Direct RNA sequencing with ONT
Drug resistance testing (HIV) using Illumina s MiSeq
Experimental setup HIV genome PRRT IN ENV whole genome
Fragmentation
Automated Nextera XT library preparation
putting things together mapping PRRT IN ENV Reference sequences
Coverage ~10K to 20K ~10K to 20K PR/RT ENV ~7500 full V3 loops
Validation isolation NGS Sanger pooling rt-pcr/ nested pcr fragmentation & indexing sequencing 400 samples (PR/RT) sequencing analysis analysis comparison
PR/RT # resistance mutations found 236 230 239 252 275 324 499
Viral load cop/ml How much virus is relevant? 1% 1%
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
Sanger vs NGS (same amplicon)
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
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
Case report: T., T.-L. *1974 TDF/FTC/RPV Resistance tests, retrospective
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%)
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
The deeptypehiv report generator Use tick boxes for visualization of resistance situation: Susceptible Limited susceptibility Intermediate Resistant
The deeptypehiv report generator Preconfigured report components available
The deeptypehiv report generator, integrated HIV-GRADE interpretation
The deeptypehiv report
The deeptypehiv report
Cost per sample calculation Illumina MiSeq (2x250) (reagents only) 25 samples per week PRRT plus Integrase plus env V3 : 3.400 nts Coverage: 20.000 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
Cost per raw Megabase
Acknowledgments Bettina Welter Kirsten Becker Anna Memmer Nina Engel Anja Laux Alexander Thielen Bernhard Thiele