Biochemical and Genetic Testing Methods and Workflows
CENTOGENE´s in-depth medical expertise is supported by the application of cutting-edge technologies. We utilize almost all of the existing testing methods in our workflows, processing every sample in-house. We adhere for each testing method to the highest international standards (CAP, CLIA, ISO) for establishment and validation.
Next Generation Sequencing (NGS) describes a variety of high-throughput sequencing methods which highly increase the possible sequencing throughput and decrease the cost per sequenced base pair. In combination, this allows us to sequence not only single fragments or genes, as has been done by Sanger sequencing for many years, but also large panels, whole human exomes (WES) or even whole human genomes (WGS) in a fast and affordable manner.
CENTOGENE´s next generation sequencing department is mainly based on the Illumina sequencing technology, the current leading technology on the market. It is based on sequencing-by-synthesis (SBS), meaning that sequencing is achieved via synthesis of a new complementary DNA strand.
Sanger sequencing and other methods
The term DNA sequencing refers to methods for determining the order of the nucleotide bases adenine, guanine, cytosine and thymine – in a molecule of DNA using a capillary sequencer.
Investigates mutations that show an increased frequency in the population. There are different hotspot mutations based on the ethnicity/ancestry of each patient (as provided by physician).
Full gene sequencing:
Analysis of the entire protein-coding regions of a gene (associated with a specific disorder ) including exon-intron boundaries.
Fragment length analysis (FLA):
Some diseases are caused by insertions/deletions which cannot be detected via classical Sanger sequencing. In these cases fragment length analysis (FLA) and/or repeat primed assays are utilized to detect the extent of the expanded repeats using a capillary sequencer.
Deletion or duplication testing:
It identifies large deletions or duplications using
Most of the lysosomal storage diseases analyzed at CENTOGENE are characterized by mutations (defects) of genes that encode enzymes or transport proteins. Lysosomal enzymes are biological active proteins that play a role in degradation of the complex molecules foreign or indigenous to the cell as part of their metabolism. The genetic defect of the respective gene is translated at the protein level (enzyme level) into an impaired or absent function. Thus, molecules which would normally be further processed or degraded by the enzyme, will accumulate – first in the lysosome, then in the cytosol and in the end in the intracellular space. Biochemical analyses can: (i.) assert the specific enzymatic activity, or (ii.) measure the levels of the substrate accumulated in the cells (biomarkers).
Measurement of enzyme activities for lysosomal storage disorders (LSDs): enzymes extracted from dried blood spots (DBS) or leucocytes convert artificial substrates into products which can be quantified using tandem mass spectrometry or via fluorimetric/spectrophotometric methods.
Measurement of biomarker concentrations in dried blood spots via tandem mass spectrometry.
Latest scientific articles - Biomarker and biochemical analysis
The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles…
Identification of a Novel Deletion in the MMAA Gene in Two Iranian Siblings with Vitamin B12-Responsive Methylmalonic Acidemia
Adenosylcobalamin (vitamin B12) is a coenzyme required for the activity of methylmalonyl-CoA mutase. Defects in this enzyme are a cause of methylmalonic acidemia (MMA). Methylmalonic acidemia, cblA type, is an inborn…
Molecular, Biochemical, and Structural Analysis of a Novel Mutation in Patients with Methylmalonyl-CoA Mutase Deficiency
Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). We found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C > G).
Which tests are based on NGS technology?
Identifies the molecular basis of a genetic disorder in an affected person by analyzing the total number of exons (= exome), the complete protein-coding region of the genome. These regions make up in total approx. 1% of the genome.
Reveals comprehensive information about the genetic composition of an individual by exhaustively covering the whole genome, thus both protein-coding and non-coding regions.
CENTOGENE’s Illumina Bioinformatics pipeline for the analysis of WES data is based on the 1000 Genomes Project (1000G) data analysis pipeline and GATK best-practice recommendations and is composed from the widely used open source software projects bwa 0.7.10,1 Picard-tools 1.138 and GATK 3.2,2; 3 snpEff 4.1,4 BEDTools 2.25.0,5 bbduk v35.x and samtools 0.1.19,6 freebayes 0.9.20, and custom-developed software (Figure 1).
How it works
Firstly, raw sequencing reads are converted into standard fast format. The quality of the sequencing reads is assessed for checking of any deviation from expected quality that would prevent from further analysis of sequencing data. Then short reads are aligned to the currently validated build of the human reference genome using BWA software with the MEM algorithm.
The alignments are converted to binary BAM file format, sorted on the fly and deduplicated without intermediate input-output-operations to temporary files to achieve maximal performance. The primary alignment files for each sample are further refined and augmented by additional information following GATK best-practices recommendations. Base Quality Score Recalibration (BQSR) is applied to improve accuracy of per base quality scores and to ensure better convergence to the actual probability of mismatching the reference genome.
To minimize false positive variant calls on genomic regions containing Insertions-Deletions (InDels), local realignment of reads around InDels is performed. Afterwards variant calling is performed on the secondary alignment files using three different variant callers. The GATK HaplotypeCaller is applied using standard parameters and if suitable limited to the target regions of capture/amplicon-based assays. In addition to GATK Haplotypecaller two other commonly used variant callers, freebayes and samtools, are applied.
Variants are annotated using Annovar7 and in-house ad hoc bioinformatics tools. Alignments are visually verified with the Integrative Genomics Viewer v.2.38 and Alamut v.2.4.5 (Interactive Biosoftware, Rouen, France).