Simple and intuitive software to analyse your sequencing data.
The Qseq is a compact, automated personal sequencing instrument. It is used to analyse up to 48 samples in terms of:
- Genotyping one or more variable positions in a DNA template (e.g. haplotypes)
- Quantifying the proportion of one or more alleles at various positions (e.g. somatic mutations)
- Quantifying the amount of methylation at specific CpG sites (epigenetics)
- Determining the sequence of a DNA template through de novo sequencing
Allelic Quantification & SNP Genotyping
Detect and quantify multiple mutations, including insertion and deletions, within a specific region of interest. Somatic mutations such as the common oncogenic NRAS, EGFR, KRAS and BRAF mutations can easily be detected to a low as 5%. The software determines the percentage of a mutation by comparing the peak height of each detected allele, from a set of possible alleles, to a set of reference samples. Various ‘control’ dispensations are automatically loaded as part of a ‘quality control’ for the run. There is also an Eco protocol for assays requiring short read lengths. The Eco protocol reduces reagent usage by half allowing a substantial cost savings.
Figure 1. Quantify up to four alleles within the one site. The software provides a simple profile of the assay, outlining the potential alleles within the variable site (clear bars) and the expected bases within the sequence (blue bars). The expected bases are used as references to help calculate the percentage of each allele present. In this example for the NRAS codon 61 oncogenic marker, only the A base was detected.
Figure 2. Determine multiple types of mutations within the one sample. Here we determined the G to C single nucleotide polymorphism and AT insertion deletion for the UGT1A1 gene. In the sample the SNP was homozygous to the wild type G-allele. The insertion of an extra AT repeat in the TATA box of the gene for one of the alleles showed us this sample was heterozygous. The added AT repeat is a diagnostic indicator for Gilberts disease.
Epigenetic modifications to DNA such as methylcytosine and hydroxymethylcytosine, which typically occur at 5’-CpG-3’ dinucleotides, play a major role in the control of gene expression and therefore cellular regulation and development. Variations in the amount of methylation have been linked to cancer, embryonic development, X-chromosome gene silencing, and cell cycle regulation. Using bisulfite converted DNA, the Qseq is capable of accurately quantifying methylation levels from 5 – 95% at multiple CpG and even non-standard methylation (CpN) sites. Sequences as long as 150 base pair can be analysed, with anywhere up to 15 or more CpG sites per read length. The software also has provision for a bisulfite control dispensation that can determine the quality of the bisulfite conversion.
Figure 3. Accurately quantify the percentage methylation at individual CpG sites. The Qseq can cover over 100 base pairs per assay, to allow quantification of many CpG sites, such as this example for the single-minded 2 (SIM2) gene promoter site. Here we analysed 7 sites across a total of 73 base pairs. The inclusion of a bisulfite conversion control also provides further feedback on the quality of the data.
De novo Sequencing
De novo sequencing on the Qseq allows for the determination of an unknown sequence, or more commonly the verification of a PCR product. Many users will find such an application useful when developing new PCR assays or as part of a QC process for a molecular diagnostic. Other uses include verification of difficult to distinguish samples from high resolution melting, verification of clones, and the detection of new mutations within the region of interest.
De novo sequencing applies a pre-determined repetitive cycle of the four nucleotides over many dispensations. The resulting peaks from the reaction are used to determine the sequence of the target by using an algorithm to calculate the homopolymer length at each positive signal dispensation. Each sequence can then be exported into programs such as BLAST to determine its identity. Sequence lengths of up to 150 base pairs are possible on the Qseq.
Figure 4. Determine or confirm fragments up to 150 base pair long. We de novo sequenced up to 87 bases of human chromosome 4.
Figure 5. Search for mutations within specific areas of your template. In this example we looked for mutations within the first half of the second exon for the oncogenic marker KRAS. The dispensation order contained a combination of known sequence, and de novo sequencing at areas of interest.