Prof. Peter Nürnberg: Functional Genomics (Cologne Center for Genomics [CCG], Institute for Human Genetics)

Next generation sequencing (NGS) methods are currently revolutionizing the field of genome
research. They enable researchers to decode DNA and RNA sequences at an unprecedented
depth and level of detail. The Cologne Center for Genomics set up cutting-edge NGS
technologies to assist all life scientists of the University of Cologne in their bio-medical research
efforts.
1) Genomics – Whole genome shotgun sequencing (WGS): Random fragments of a
complete genome are sequenced using PCR-free library preparation. Standard pipelines for
alignment against a reference sequence and variant detection are available. De novo
sequencing: random fragments of up to 10kb original size are sequenced. Standard
pipelines include de novo assembly and annotation. Non-sequencing-based genome
mapping and linked-read technologies are used to improve the results. Structural
variations, e. g. in cancer cells, can be analyzed with similar methods and can be validated
using DNA combing technology. Targeted resequencing, including whole-exome
sequencing (WES): Random fragments of a selected genome region are sequenced using
various enrichment technologies. Standard pipelines for alignment against a reference and
variant detection are available. ATAC-seq and HiC methods are used to detect changes in
chromatin conformation.
2) Transcriptomics – RNA-seq: Reverse transcribed cDNA fragments from total RNA of any
organism are sequenced. Standard analyses include alignment against a transcript database,
detection of splice sites and gene expression estimation. Different approaches exist for
different needs like mRNA-seq using Poly-A selection, total RNA-seq using hybridization
based ribo-depletion protocols or 3´mRNA-seq for simple and cost effective expression
profiling. Low-input RNA protocols are available for 10pg-10ng and 500pg-100ng starting
material. Single cell RNA sequencing (scRNA-seq) is supported with two different
platforms based on either nano-dispensing or micro-droplet technologies.
3) Epigenomics – ChIP-seq/RIP-seq: DNA or RNA fragments extracted by chromatin
immunoprecipitation are sequenced. Standard analysis pipelines include alignment against a
reference genome, detection of binding sites (peaks) and normalization against a control.
Small RNA-seq: Reverse transcribed cDNA fragments of size-selected RNA molecules are
sequenced. Standard analyses include expression comparison, sequence variation
assessment and prediction of novel micro RNAs. WGBS: Bisulphid treated DNA is
sequenced to allow for detection of DNA; ethylation in any organism.
Internal resources: The core of the facility consists of Illumina’s HiSeq4000, HiSeq2000, Miseq
sequencers. Further Illumina iScan and Affymetrix Gene Titan array platforms are in place.
Diverse robotic systems from Beckman, Hamilton, Tecan and Agilent are in use for automated
protocols. ABI 3730 Capillary Sequencers Applied Biosystems are used for validation/segregation
studies. Further equipment comprises numerous PCR cyclers and qPCR divices from Applied
Biosystems, Diagenode’s Bioruptor, 2200 Tape Stations from Agilent, PeqLap’s NanoDrop, and
Invitrogen’s Qubit. For cfDNA studies the Qiagen PSQ96HS (Pyrosequencer) and Applied
Biosystems’ QuantStudio® 3D Digital PCR System are available. Dedicated equipment for
genomic mapping /SV detection includes BioNano’s Irys System and the Fiber Vision System
from Genomic Vision. For scRNA-seq the ICELL8 System from Wafergen and the Chromium™