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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, three MiSeq systems and a PacBio Sequel II.

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, despite the falling prices. WGS approaches can be divided 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 II HiFi 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 re-sequencing, we offer two different library preparation protocols that differ in their mode of initial DNA fragmentation (enzymatic vs. mechanical shearing). For low input protocols or special input like FFPE-DNA or cfDNA for Illumina libraries, please take contact ccga@ikmb.uni-kiel.de.

    Offered Library types (PDF):
    • Illumina DNA Prep (tagmentation)
    • Illumina DNA PCR-Free Prep (tagmentation)
    • TruSeq DNA Nano
    • (multiplex) SMRTbell - HIFI, low input and ultra-low input

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 annotation by providing exon sequence information, PacBio full length transcript can provide isoform information or information on fusion genes, 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 and their input requirements. Please contact us ccga@ikmb.uni-kiel.de for highly degraded samples and/or ultra low input protocols. We also offer single-cell RNA-sequencing (see below).

    Offered Library types (PDF):
    • TruSeq stranded mRNA/Illumina Stranded mRNA Prep
    • TruSeq stranded total RNA
    • Illumina Stranded Total RNA Prep with Ribo-Zero Plus (bacterial depletion)
    • NextFlex small RNA v3
    • NextFlex Combo-Seq kit (mRNA and miRNA in one protocol)
    • 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). For panel seq. please contact us in advance via ccga@ikmb.uni-kiel.de.

    Offered Library types (PDF):
    • Illumina DNA Prep with Enrichment IDT xGen Exome
    • HLA typing

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

6. Single Cell Transcriptomics

We use the 10x Genomics chromium controller to prepare single cell expression tags from cell suspensions. Single cell libraries come in different flavors, however: all of them require > 95 % live cells, so for people from outside of Kiel, it might be best to submit ready-to-sequence libraries. Please contact us ccga@ikmb.uni-kiel.de in case you need help finding a solution.

    Offered Library types (PDF):
    • 3' expression tags
    • 5' expression tags
    • V(D)J sequencing
    • T and/or B-cell specific expression tags

News

Mar. 03, 2022
5th DFG Sequencing Call now open ...

Mar. 03, 2022
limited capacities ...

Mar. 02, 2021
4th DFG Sequencing Call now open ...

Feb. 11, 2021
Temporary stop for new projects until end of March ...

Feb. 11, 2021
CCGA is sequencing SARS-Cov2 strains ...

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