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Vienna 16/05/2017
Presented by Antonio Alonso on behalf of the DNASEQEX ConsorAum
61% 18%
12% 6% 3%
Type of Respondent Organiza3ons
Police laboratories Ministry of JusAce University
Ministry of Health Private Agency
33 ENFSI DNA WG laboratories from 25 countries
ParAcipaAon: 54% of all ENFSI DNA Labs (53 Member InsAtutes and 8 Associated Members)
32
30
30
23
29
5
0 5 10 15 20 25 30
Forensic DNA analysis from traces in criminal cases
UnidenAfied Human Remains and Missing Persons
ContribuAon To NaAonal DNA Databases
Parentage TesAng
Research and ValidaAon
Others
Number of Labs
Indicate the areas of forensic DNA applica3ons performed in your laboratory
52% 48%
Does your laboratory have a Massively Parallel Sequencing instrument?
YES
NO 61%
39%
Does your laboratory have a Massively Parallel
Sequencing instrument or Will your laboratory purchase a MPS instrument in the next
18 months?
Yes
No
3.6 3.2 3
2.7 2.7
1.95 1.9 1.9
0 1 2 3 4 5
No available funding
Methodology is too expensive
WaiAng unAl MPS applicaAons are more fully developed
Instruments are too expensive
No experAse available to perform bioinformaAcs
No experAse available to perform tesAng
No lab space available to perform tesAng
No need for the technology (CE addresses the vast majority
What are the reasons for currently NOT purchasing a MPS instrument?
Responses from 16 labs ranked in order of priority from 1(lowest) to 5 (highest)
41%
41%
18%
Please indicate the methods used for DNA library prepara3on, DNA template
prepara3on, and cloning
Manual Manual and Automated Automated
10
7
4 3
How many of the following MPS instruments does your laboratory have?
Illumina MiSeq
Ion Torrent PGM
Ion Torrent S5
Others (1 lab)
2 Hamilton & 1 Beckman placorms
for Illumina Workflow
7 Ion Chef™ for Thermofisher Workflow
AutomaAon (somehow) leads us away from scienAfic knowledge • User guides on automated MPS Technology should be comprehensive, describing in detail the different
processes and steps involved in DNA library and DNA template preparaAon (Genomic PCR, Barcoding by ligaAon or PCR, Library equalizaAon and purificaAon, Solid-‐PCR of libraries, Enrichment, Chip Loading,..).
• Informa3on on relevant parameters (such as PCR-‐Setup and pipelng parameters, PCR parameters, PurificaAon parameters,…) should also be included as it is needed for the validaAon and the interpretaAon of the data.
10
9
2
6
0 2 4 6 8 10 12
Illumina UAS
Torrent Suite Sonware
Converge (Beta version)
Open Source Sonware
Please indicate the soYware used for Sequence Assembly and Data Analysis
Open Source Sonware
• STRait Razor v2.0 • FDSTools
72%
14% 14%
How do you store MPS data generated in your laboratory?
In-‐house server
Instrument Manufacturer Owned Cloud
External Server
DNASeqEx Sonware ValidaAon
Data analysis of MPS-‐STR panels by Commercial SoYware (UAS and TSS/Converge)
Improvements to recover full informa3on from MPS sequence data
• Sequence coverage data on forward and reverse strands
• STR array sequence data + flanking regions sequence data
• Genome coordinates of the sequence read start and end points
• Comprehensive and global STR nomenclature system (backward compaAbility with STR CE data)
• Global output file for internaAonal exchange
14
11 9
11
8 8 8
3 3 2
19 18
17 15 15
14
11
8 8
3
Please check for which category or categories of Forensic DNA markers MPS is currently used or being evaluated in your lab and
for which ones will be developed in the next 18 months
Number of labs in December 2016 Number of labs in 2018
> 75%
Innsbruck Berlin Madrid
DNASeqEx ValidaAon studies
STR Kits Valida3on Studies
• ForenSeq™ DNA Signature Prep Kit (Panel A: 27 Autosomal STR, 24 Y-‐STR, 7 X-‐STR, and 94 SNPS) • Precision ID GlobalFiler® Mixture ID Panel (29 autosomal STRs, 1 Y STR, 42 SNPs, 2 Y-‐ SNPs,36 microhaplotypes clusters, Amelogenin and the Y indel rs2032678) • Precision ID GlobalFiler® STR ID Panel (29 autosomal STRs, 1 Y-‐STR, Amelogenin)
• AnalyAcal, InterpretaAon and Stuter Thresholds
• Concordance to CE • Reproducibility • SensiAvity • Mixture studies • PopulaAon studies • Forensic samples
DNASeqEx ValidaAon studies
STR Kits Mee3ng Presenta3on
• ForenSeq™ DNA Signature Prep Kit (Panel A: 27 Autosomal STR, 24 Y-‐STR, 7 X-‐STR, and 94 SNPS)
STR Kits Mee3ng Presenta3on
• Precision ID GlobalFiler® Mixture ID Panel
(29 autosomal STRs, 1 Y STR, 42 SNPs, 2 Y-‐ SNPs,36 microhaplotypes clusters, Amelogenin and the Y indel rs2032678) • Precision ID GlobalFiler® STR ID Panel (29 autosomal STRs, 1 Y-‐STR, Amelogenin) Pedro Barrio
Petra Müller Do not miss it.
Today 15:20–16:20
3.7
3.5
3.4
3.1
2.8
2.3
2
0.4
0 0.5 1 1.5 2 2.5 3 3.5 4
Lack of nomenclature and reporAng standards
NaAonal DNA Databases cannot accommodate the new data
Not sufficient populaAon data
There is a lack of an adequate naAonal legislaAon framework
No specific validaAon guidelines available
No specific proficiency tests available
Not demonstrated to be reliable
Others
What do you think are the main scien3fic and legal challenges for the implementa3on of MPS in Forensic Gene3cs?
Responses from 33 labs ranked in order of priority from 1(lowest) to 5 (highest)
Nomenclature and Na3onal DNA Databases compa3bility
ü Simple STR nomenclature systems (numbers and le-ers) are easy to communicate and therefore preferred for rou7ne exchange of STR data between analysts and stakeholders
ü However, the transla7on process will have to be managed by a centralized nomenclature commission to avoid ambiguous or imprecise allele names being adopted, or assigning different names to iden7cal alleles.
ü It has been suggested that an online system could be used that is curated by a nomenclature commission, which would be responsible for new allele designa7ons upon valida7on of the observed sequence varia7on.
ü Criteria for the valida:on of sequence varia:on and its comparison with exis7ng variants need to be defined in more detail
Popula3on studies on STR sequence varia3on
A publicly available, centrally curated online allele frequency database and
quality control plaHorm
STR sequence data in STRidER
The storage of nucleo:de strings (text strings) in FASTA-‐like format has been iden7fied as the core framework to permit soIware-‐aided alignment and transla7on between STR nomenclatures (MPS & CE). The exis7ng architecture of STRidER allows for the implementa7on of nucleo7de sequence strings (as kept in EMPOP) and thus is fully compa:ble with the QC of popula:on data generated by MPS.
DNASeqEx Present and Future Plans
SYSTEMATIC EVALUATION OF MASSIVELY PARALLEL STR SEQUENCING DATA MiSeq FGx benchtop sequencer (Illumina Inc., CA) or the Ion S5 sequencing system (Thermo Fisher ScienAfic Inc., CA) LIMS INTEGRATION OF NEW MASSIVELY PARALLEL SEQUENCING WORKFLOWS IN FORENSIC GENETICS ConfiguraAon of a commercial LIMS system (LabWare Inc., DE) to fit the automated workflow provided by the Ion Chef / Ion Torrent S5XL placorms and the Torrent Server Suite / Converge sonware packages. NOMAUT – NGS STR Nomenclature for Forensic Gene3cs Web Self-‐maintaining NOMenclature AUThority sonware
Acknowledgement
ParAcipaAng Laboratories
The Faculty
InsAtute of Legal Medicine, Medical University of Innsbruck. Austria
InsAtute of Legal Medicine and Forensic Sciences, Charité –Universitätsmedizin Berlin. Germany
Center for Human IdenAficaAon at the University of North Texas Health Science Center. EEUU
NaAonal of Toxicology and Forensic Sciences. Madrid Department. Spain
Steffi Köcher
Speaker was provided travel and hotel support by Thermo Fisher ScienAfic for this presentaAon, but no remuneraAon When used for purposes other than Human IdenAficaAon or Paternity TesAng the instruments and sonware modules cited are for Research Use Only. Not for use in diagnosAc procedures. Thermo Fisher ScienAfic and its affiliates are not endorsing, recommending, or promoAng any use or applicaAon of Thermo Fisher ScienAfic products presented by third parAes during this seminar. InformaAon and materials presented or provided by third parAes are provided as-‐is and without warranty of any kind, including regarding intellectual property rights and reported results. ParAes presenAng images, text and material represent they have the rights to do so.
Acknowledgements
This project has been funded with support from the European Commission (grant HOME/2014/ISFP/AG/LAWX/4000007135 under the Internal Security Funding Police programme of the European Commission-‐Directorate General JusAce and Home Affairs). This publicaAon reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the informaAon contained therein.