Automation in genomics High-throughput management of biological samples and nucleic acids. Valentina Gualdi Operational Scientist PGP

Transcription

1 Automation in genomics High-throughput management of biological samples and nucleic acids Valentina Gualdi Operational Scientist PGP OVERVIEW PGP workflow and robots set-up Sample preparation Sample re-array Worklist Quantification and normalization of nucleic acids Magellan Software Conclusions 2 1

2 PGP general workflow Laboratory 1: Sample aliquoting from original format to barcoded 96-well plates (Tecan-Freedom EVO 100) Nucleic acids extraction, quantification and normalization (Tecan-Freedom EVO 150) PCR, RT-PCR and RealTime PCR set-up (Tecan-Freedom EVO 75) Laboratory 2: PCR purification (Tecan-Freedom EVO 150- TeMO) Genotyping reactions set-up (e.g. sequencing, SNapShot, SNPlex) (Tecan-Freedom EVO 150- TeMO) Capillary electrophoresis (3730 DNA Analyzer) Quantitative PCR (7900HT) 3 WORKSTATION 1 - Freedom EVO 100 Sample preparation PCR set-up Liquid handling arm (LiHa): 4 channel with disposable tips; liquid detection and clot detection system Robotic manipulator (RoMa arm): to move labwares (microplates, lids, deepwell ) between the worktable and peripheral modules Positive Identification System (PosID): robotic barcode scanner to identify and monitor labware carrier positions and to decode different types of barcodes on microplates, tubes or card 4 2

3 WORKSTATION 2 - Freedom EVO 150 NA extraction, quantification and normalization LiHa: 8 channel with disposable tips RoMa arm 3 Hotels: for storage of different types of labwares (MPT, Deep-well ) Barcode reader: to scan barcodes on labware during transfer actions Te-VacS: vacuum separation module for fully automated NA extraction (96-well plate) Thermo-shaker: for the automation of incubation step Tecan Genios reader: for DNA and RNA quantification and normalization via fluorescence and absorbance measurement 5 WORKSTATION 3 - Freedom EVO 150 TeMO Sequencing set-up, PCR/ sequencing clean-up LiHa: 8 channel, fixed tips, low volume option RoMa arm Te-MO, 96-channel head: multichannel pipetting option with 15 position deck, for complex assays (e.g. SNPlex) development and high-throughput analyses Carousel HS with barcode option: for fast access to different types of labwares, sample tracking, highthroughput Power washer 384: high performance washer for 96 and 384 MTP SNPlex System Automation system 6 3

4 WORKSTATION 1 - Freedom EVO 100 Sample preparation (1) Starting material: Every kind of liquid samples (serum, blood, DNA/RNA, homogenized tissues, bacterial cultures) Different liquid classes clot detection system Different volumes (Robot set-up) Single or multi-dispensing Different kind of labwares: vials (eppendorf, cryotubes, vacutainers) or plates (96 or 384MPT, deep-wells) Different carriers (standard or custom) Barcodes (POS-ID) 7 WORKSTATION 1 - Freedom EVO 100 Sample preparation (2) - 8 4

5 WORKSTATION 1 - Freedom EVO 100 Sample preparation Blood aliquoting (1) Script for aliquoting blood samples from 96 vacutainers with barcodes to 2 differents plates: 1stock plate and 1 extraction plate POS-ID to read barcodes and save information 9 WORKSTATION 1 - Freedom EVO 100 Sample preparation Blood aliquoting (2) 10 5

6 WORKSTATION 1 - Freedom EVO 100 Samples re-array Creation of work-plate from several different stock plates 11 WORKLIST (1) Possibilty to create worklist: CSV, excel Kind of data: Source labware (e.g. MPT, vial, vacutainer ), source position (e.g. well ID), volume, destination labware, destination position Very simple, safe and rapid way to create script Possibilty to export files of each labware Source labware DNA6 DNA6 DNA7 DNA7 DNA8 DNA8 Source position volume Destination labware Destinatio position

7 LOAD WORKLIST source labware;source position;volume;destination labware;destination position DNA6;1;10;;1 DNA6;80;5;;2 DNA7;5;12;;3 DNA7;23;23;;4 DNA8;35;2;;5 DNA8;96;15;;6 13 EXECUTE WORKLIST 14 7

8 WORKSTATION 2 - Freedom EVO 150 Samples re-array Wide flexibility Particular kind of re-array for specific applications Ex. 2D pooling Tilling 15 WORKSTATION 2 - Freedom EVO 150 quantification and normalization of NA Tecan Genios reader: for DNA and RNA quantification and normalization via fluorescence and absorbance measurement Pico-green for DNA quantification just double-strand DNA as little as 25pg/ml dsdna solution very dynamic range (from 25pg/ml to 1000ng/ml) Absorbance at 260nm (A 260 ) relative contribution of nucleotides and single-stranded nucleic acids interferences by contaminants less sensitivity (A = 5µg/ml dsdna solution) Magellan software to read plates Used of Magellan through Freedom EVO Possibility to save standard curve For precise measurement necessity to create and read standard curve for each plates Excel macro to normalize DNA concentrations after measurement 16 8

9 WORKSTATION 2 - Freedom EVO 150 Magellan Software (1) Wizard to easily create method to perform quantification 17 WORKSTATION 2 - Freedom EVO 150 Magellan Software (2) 18 9

10 WORKSTATION 2 - Freedom EVO 150 Magellan Software (3) Export results as: Raw data Single concentration Std curve Transformed data 19 WORKSTATION 3 - Freedom EVO 150 Samples re-array Possibility to create high number of child plates from mother plate Very quickly with Te-Mo 96 samples moved all together Possibility to quickly create work dilution Te-Mo used also to create pool of samples (ex. Microsatellite run plates) Fixed tips 20 10

11 CONCLUSIONS ADVANTAGES OF AUTOMATION OF BIOLOGICAL SAMPLES AND NUCLEIC ACIDS MANAGEMENT Traceability barcodes export files txt, excel different labwares coordinates and volumes log-files and error-files High-throughput management of samples Precision REproducibility, repeatibility, low rate of errors 21 11