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Next-Generation-Sequencing

Next-Generation-Sequencing (NGS) is a high-throughput method for massive, parallel sequencing of millions of nucleotide fragments. A NGS experiments needs a carefully evaluated study design, good quality source material (DNA or RNA), a so-called library preparation, the sequencing run to generate big datasets and bioinformatic / statistician expertise to extract meaningful biological results from these data.
In our institute, we offer to do the library preparation process as well as the NGS sequencing on the basis of a scientific collaboration. Currently, the fleet of NGS sequencing devices in our institute comprises two Illumina NovaSeq 6000, two HiSeq 3000/4000, one NextSeq 500, two MiSeq systems and a PacBio Sequel.

NGS Library Types

1. Whole-Genome Sequencing

Whole-Genome-Sequencing (WGS) is an approach to determine the complete DNA sequence of an organism's genome, consisting of its chromosomal DNA as well as DNA contained in mitochondria and, for plants, in its organelles. While WGS allows the researcher to have a comprehensive look at the genome, its biggest drawback is its comparatively high price tag. WGS approaches can be devided into de-novo-sequencing projects and re-sequencing projects.

    1.1 De-novo-WGS

    For de-novo genome sequencing, long reads are beneficial. In our lab, we recommend the PacBio Sequel for small to medium genomes, and for large genomes, a hybrid approach with Illumina short reads and PacBio long reads can improve assembly results.

    1.2 WGS-Re-Sequencing

    WGS-Re-Sequencing is considerably cheaper than de-novo-WGS, since it can be achieved by short reads alone if a high-quality assembled reference genome is present. For WGS resequencing, we offer two different library preparation protocols that differ in their mode of initial DNA fragmentation (enzymatic vs. mechanical shearing).

    Offered Library types (PDF):
    • Nextera DNA Flex
    • TruSeq DNA Nano
    • (multiplex) SMRTbell

2. Transcriptome Sequencing

Transcriptome Sequencing (RNAseq) sequences the expressed RNA transcripts from a source tissue. Transcriptome data has many uses. It can guide de-novo-genome assembly by providing exon sequence information and it can provide transcript counts for differential gene expression (DGE). We currently offer a variety of different transcriptome library preparation workflows that differ in their target RNA sub-populations. We also offer single-cell RNA-sequencing from single-cell-suspensions.
    Offered Library types (PDF):
    • TruSeq stranded mRNA
    • TruSeq stranded total RNA
    • TruSeq Exome (for FFPE and low input samples)
    • NextFlex small RNA v3
    • NextFlex Combo-Seq kit (mRNA and miRNA in one protocol)
    • Chromium single-cell RNAseq
    • PacBio IsoSeq

3. Targeted Sequencing

Targeted Sequencing (syn. Enrichment Sequencing, Panel Sequencing) is a method to reduce the complexity (and therefore the costs) of Whole-Genome-Sequencing (WGS) libraries. The most prominent example is Whole-Exome-Sequencing (WES), which enriches exonic regions of the genome by hybridizing them to pre-designed oligo baits and capturing the resulting duplex with magnetic streptavidin beads. Instead of WES, also smaller oligo bait sets can be used, e.g. to enrich only for disease-relevant genes of interest (so-called Panel-Seq), or an amplicon based approach can be done (please refer to the section microbiome sequencing for more details on amplicons).
    Offered Library types (PDF):
    • Nextera IDT xGen Exome
    • HLA typing
    • multiplexed SMRTbell + panel

4. Methylome Sequencing

Methylation of cytosines is an epigenetic feature of eukaryotic genomes and is fundamental for cellular differentiation processes including transcription control. Interestingly, while methylation patterns can be inherited, they can also change during the lifespan of cells and tissues and is susceptible to e.g. diet or other environmental influences. In our institute, we offer methylome sequencing in the form of Reduced-Representation-Bisulfite-Sequencing (RRBS) and Whole-Genome-Bisulfide-Sequencing (WGBS). For bacteria, A-methylation is present as well, which can be detected directly by PacBio sequencing without the need for base conversions.
    Offered Library types (PDF):
    • RRBS
    • NEB Em-Seq (enzymatic conversion corresponding to bisulfite treatment)
    • multiplexed SMRTbell

5. Microbiome Sequencing

The microbiome (i.e. the genomic content of the host-associated microorganisms, comprising bacteria, archaea, fungi, protists and viruses) is considered to be an important factor for the immunologic, hormonal and metabolic homeostasis of host organism. In our institute, we offer taxonomic profiling via amplicon sequencing (e.g. 16S amplicon sequencing) or taxonomic and functional profiling via metagenome shotgun-sequencing.
    Offered Library types (PDF):
    • Shotgun Metagenome Sequencing (Nextera DNA Flex)
    • Bacterial 16S amplicon V1/V2
    • Bacterial 16S amplicon V3/V4
    • Archaeal 16S amplicon
    • Fungal ITS amplicon
    • for self-made other amplicons, selfmade libraries or PacBio amlicon-sequencing, please contact us via ccga@ikmb.uni-kiel.de

News

Oct. 26, 2020
Follow us on Twitter @CcgaKiel ...

Sep. 09, 2020
Turn around time momentarily increased: 12 weeks ...

Mar. 30, 2020
3rd DFG Sequencing Call: new deadline 9.9.2020 ...

Mar. 16, 2020
Limited service due to Corona ...

Dec. 12, 2019
Christmas break 19.12.2019 - 06.01.2020 ...

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