Sequencing Unit

CNAG-CRG

HEAD OF THE UNIT: Marta Gut

LABORATORY MANAGERS: Julie Blanc, Katja Kahlem, Lidia Agueda

LABORATORY TECHNICIANS: Marta Lopez, Ana Gonzalez, Pili Herruzo, Maite Rico (maternity leave from February to September), Caterina Mata (maternity leave from April), Laetitia Casano, Aurora Padron, Yasmina Mirassou (maternity leave from February), Giulia Lunazzi (maternity leave until May), Regina Antoni, Ester Bonastre, Nuria Aventin, Sonia Villanueva Hernandez (from May to November), Caterina Carbonell (from May), Carme Fabregat (from August).

SPECIALIZED TECHNICIANS: Javier Gutierrez, Silvia Speroni

QUALITY MANAGER: Lidia Sevilla

SINGLE CELL GENOMICS

TEAM LEADER: Holger Heyn

POSTDOCTORAL FELLOWS: Amy Lauren Guillaumet (until July), Catia Moutinho (from July), Gustavo Rodriguez, Giovanni Iacono (from March)

PhD STUDENTS: Atefeh Lafzi, Davide Polizzi (until September)

DATA ANALYST: Elisabetta Mereu

Summary

The excellent quality is an added value the CNAG-CRG Sequencing Unit is providing to the Spanish and international scientists for each project. The Sequencing Unit is positioned uniquely within Europe with its scope and mission to also provide reliable and valid NGS tests for clinical research and for guiding clinical decisions. In 2017, thousands of libraries were prepared and sequenced following strict SOPs (Standard Operating Procedures) with assured consistency, accuracy and precision by the quality management system of ISO 9001:2015, re-certified by an external annual audit by the SGS (Société Générale de Surveillance) and by the technical competence re-certification through the external annual audit by the ENAC (Entidad Nacional de Acreditación) for ISO/IEC 17025:2005 accreditation.

The CNAG-CRG Sequencing Unit comprise five teams – the Biorepository, the Sample Preparation team, the Production team, the Support team and the Single Cell team who work side by side around LIMS (Laboratory Information Management System) supported by the database development team. The sequencing equipment and the auxiliary instrumentation is kept state-of-art of a modern genome center. In 2017, the Sequencing Unit incorporated an Illumina HiSeq3000 sequencer in addition to other sequencers – HiSeq4000, two HiSeq2500s, MiSeq and three Oxford Nanopore Technologies (ONT) MinIons. In order to keep pace with the scaling-up of the sequencing operation we introduced in the Biorepository a Gilson Pipetmax robot, and the Sample preparation team accommodated to the increased numbers of samples processed by implementing a Mantis (Formulatrix) liquid handling system.

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Research Projects

  • Defining a pipeline for input material quality control parameters for ONT MinION MkI sequencers:​
    • Implementing Pippin Pulse as a sample quality instrument for assessment of the DNA integrity
    • Assessing quantity, contamination and purity of the nucleic acids using numerous Nanodrop parameters
  • Expanding protocol portfolio of sample preparations for ONT MinION MkI sequencers
    • 1D2 and Direct RNA sequencing kit
    • Development of RNA-seq for ONT MinION MkI sequencers combining SMARTSeq2 and 1D protocols
  • Implementing HMW DNA extraction for long read sequenicng
  • Introducing the new MANTIS® Liquid Handler (Formulatrix)
    • Miniaturization of Kapa Hyper Prep protocol for Illumina sample preparation
  • Implementation of automated protocol for B-CAST Truseq Amplicon on different liquid handlers platforms
  • Implementation of automated protocol for TruSeq Stranded mRNA on different liquid handlers platforms
  • Extending quality control for plant DNA with setting up PCR with universal/plant specific primers of ITS region of nuclear ribosomal DNA
  • Implementation of Pipetmax (Gilson) for sample processing automation in 96-well plates for QC protocols and aliquot preparation
  • Benchmarking of three capillary based electrophoretic systems (2100 Bioanalyzer (Agilent), Fragment Analyzer (AATI) and 4200 Tapestation (Agilent))
  • Benchmarking TSCA LI panel (TruSeq Custom Amplicon Low Input Kit) for B-CAST (Breast CAncer Stratification) consortium
  • Setting up automated pipeline for TSCA LI panel – version1 and version2
  • Extensive comparison of kits Kapa Hyper Prep, Kapa Hyper Plus and NEB Ultra II for B-CAST consortium
  • Benchmarking Illumina TruSeq Exome Capture and TruSeq EPIC Methyl Capture
  • Improvements of the protocol Stranded mRNA-Seq library construction – Illlumina TruSeq on the Sciclone robot
  • Preparation and passing the re-certification audit of ISO9001:2015 (including the transition to las revision of the standard).
  • Preparation and meeting the requirements for ISO10725:2005 maintenance audit and the extension of the accredited scope.
  • Incorporation of four new protocols to the technical annex (SOP-143, SOP-144, SOP-145, SOP-147)
  • Testing and calibration performed using standard methods for expansion of protocols for ISO 9001 and 17025 audit
    • Kapa stranded mRNA (Roche)
    • SMARTseq2
    • KAPA RNA Hyper Prep Kit with RiboErase (Illumina)
  • Enlarging the protocol portfolio of single-cell transcriptomics applications
  • Establishing of automated protocols for single-cell SMARTseq
  • Implementation of Seq-Well, a nanowell-based single-cell capture technique
  • Introducing TCR genotyping from single cells
  • Evaluation of single-cell whole-genome DNA amplification methods
  • Implementing RNA-seq protocol for low input protocol for low quality samples