P01-32_170222
LIMS CONFIGURATION TO FIT NEW MASSIVELY PARALLEL SEQUENCING WORKFLOWS IN FORENSIC GENETICS Pedro A. 1
Introduction
1 Barrio ,
Pablo
1 Martin ,
The DNASEQEX Consortium, Antonio
1 Alonso
Biology Service of the National Institute of Toxicology and Forensic Sciences (NITFS), Department of Madrid (Spain),
[email protected]
Massively parallel sequencing (MPS) platforms allow the simultaneous analysis of thousands of short DNA fragments, of up to hundreds of samples. In this context, their use on Short Tandem Repeat (STR) DNA profiling is promising in the field of Forensic Genetics. The DNASEQEX project (DNA-STR Massive Sequencing & International Information Exchange) seeks to explore and promote the implementation of MPS-technology for DNA profiling and international exchange. Within the framework of this project, we have developed the configuration of a commercial LIMS system (LabWare Inc., DE) to fit the automated workflow provided by the IonChef / Ion S5XL platforms and the Torrent Server Suite / Converge software packages (Thermofisher Inc., CA) for the MPS analysis of STR markers in the forensic lab. Laboratory Information Management Systems (LIMS) are valuable tools in the forensic genetics laboratory, streamlining sample and analysis traceability, batching of multiple samples, integration and data transfer with genetic analyzers, monitoring of test results, DNA data exchange with National DNA databases, and flexible reporting. This poster highlights the most important LIMS tools developed for data export, data capture, and tracking of information all along the process of library preparation, library quantification, DNA template preparation, Ion S5 sequencing, and expert Converge analysis.
Library Preparation
LIMS Samples were electronically logged into the LIMS and ion-coded using a MPSAnalysis of 4 components: MPS Kit, IonCode Kit, Barcode, and well position. After completing the data of the 4 components, a MPS batch (containing 8 samples for IonChef) was created and a file was generated and electronically imported by the TSS. A Composite Sample (the “Library sample”), containing 8 original samples, can be also created in LIMS to receive the TaqMan quantification data (in duplicate) from the HID software.
TORRENT SERVER SUITE
Library Preparation (Automated workflow) Target PCR Barcode Ligation Library Purification
S5 Sequencing & Converge
Template Preparation
Library Quantification
Normalization A Library Quantification batch was created with all those library composite samples that require quantification. A TXT file (components: Text ID, Sample Number, Test Number and Task) is generated and imported by the HID software.
HID Software
Library Quantification
Ion Library TaqMan® Quantitation Kit
When quantification was finished, the ABI7500 quantification data (CSV format) was captured by the LIMS (components: Well position and DNA concentration).
Once the libraries have been prepared and adjusted according to the concentration obtained, a LIMS run batch was generated to send any required Sample Set to the TSS (CSV file) to start the DNA template preparation at the Ion Chef and the subsequent sequencing run at the Ion S5XL.
TORRENT SERVER SUITE
Template Preparation Emulsion Preparation emPCR ISP Recovery (Enrichment)
Chip Load
At present, we are configuring information exchange between LIMS and Converge. LIMS would accept all output files generated by Converge including CMF CODIS files, and more informative XLSX files containing STR sequence data including flanking SNPs.
LIMS would use the imported Converge data to generate compatible output files for exchange to any other databases or MPS-STR projects: STRidER, NOMAUT, STRaitRaizor, CODIS,...
CONVERGE
Converge version 2.0 is a commercially available software that meets the ISFG recommendations on STR nomenclature, including the analysis of SNPs in the flanking regions of STRs.
Ion S5 STR Sequencing & Expert analysis by Converge
Raw and quality MPS chip data were generated by the TSS. The Genotyper plug-in was used to create a file for Converge import.
Conclusions The developed LIMS configuration provides the laboratory with the tools required for setting up automated MPS workflows, helping to efficiently and accurately acquire and manage MPS data in the forensic genetics laboratory, integrating different instruments and software into an automated workflow architecture. The system is flexible enough for future introduction of new equipment, procedure, and / or software to complement or improve the workflow. 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 Justice and Home Affairs). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. The authors would like to thank members of LIMS Administrators Team of the General Subdirectorate of New Technologies of Justice (SGNTJ) of the Ministry of Justice (Spain) for their helpful technical support. The authors would like to thank Thermo Fisher Scientific for support. The authors declare no conflict of interest. Follow us on https://www.researchgate.net/project/DNASEQEX
