The right answers to frequently asked questions
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What is the data handling process, including QIAGEN’s Ingenuity Variant Analysis?
All raw data are analysed and converted into a vcf file, which is uploaded directly to QIAGEN’s web-based software platform. Log-in data to your account will be provided by Eurofins Genomics.
Furthermore, the alignment file (BAM), the SNP and InDel tables (vcf and tsv) and the Data Analysis Report (pdf) from Eurofins Genomics will be delivered in the formats indicated via your online account (see below).
Please note: Customers must accept the QIAGEN End-User License Agreement before access will be granted to the Variant Analysis product or to the results generated by this product.
How long is my data accessible at my QIAGEN account?
The license grants you initial access to the analysis software for 6 months. After that period you have the option of renewing the license, but you may only purchase a renewal offline – please contact us.
Where do I get my results, if I only choose the optional BioIT?
All raw data as well as the analysed data can be downloaded via your secured online account.
What coverage should I use?
For SNP calling in heterozygote organisms we recommend at least 30x coverage. A higher coverage will increase the confidence of SNP calling. Confidently discovering genetic variants in inhomogeneous samples, such as DNA extracted from normal and tumour cells, requires higher overall coverage. The amount of data needed to reach a certain coverage on the DNA of interest depends on the ratio of normal-to-tumour DNA. Please note that due to varying efficiency of the enrichment baits, the targeted regions are not covered evenly (see below).
How do you handle PCR duplicates?
PCR duplicate rates are directly correlated to the quality and amount of starting material provided. Library preparation with less than the recommended amount of DNA requires additional PCR cycles to generate enough material to load the sequencer. Hence the PCR duplicate rate is dependent on the sample and cannot be influenced by Eurofins Genomics.
Duplicates are not excluded from the calculation of the average on-target coverage. Based on our experience with the sequencing of human samples, we typically obtain PCR duplicate rates of approximately 5% for high-quality DNA.
If further bioinformatics are ordered (e.g., SNP identification), only one copy of the duplicate read pair is kept in the alignment. The rest are excluded from further analysis to prevent any bias.
Which starting material should I send? In what quantities?
For optimum results we require at least 100 ng double-stranded, purified, high-molecular-weight, RNA-free DNA (concentration ≥ 1 ng/µL; OD 260/280 ≥ 1.8; OD 260/230 ≥ 2.0). For DNA from FFPE samples we need at least 200 ng of DNA.
Contamination by DNA from other species (> 3%; especially from closely related species) might interfere with the enrichment of the human DNA and also reduces the total amount of human DNA in the provided sample. Whole-genome amplified (WGA) DNA or DNA from archived tissue, such as Formalin-Fixed Paraffin Embedded (FFPE) samples, is usually accepted. Please contact us if you need any details.
What do you mean by overlapping reads?
The fragmentation of genomic DNA according to the Agilent SureSelectXT protocol and subsequent downstream processing produces a Gaussian distribution of DNA fragments with different lengths. A small percentage of the resulting library fragments have an insert size below 250 bp. Sequencing those library fragments with 150 bp paired-end reads will generate partially overlapping reads that cover the same bases and hence create an artificial doubling of coverage at those positions. To assure accurate analyses, those bases are excluded from further downstream analyses, such as single-nucleotide variant and insertion and deletion detection. Furthermore, Eurofins Genomics is continuously optimising its protocols to minimise the number of overlapping bases.
What is the quality of the data?
When sequencing human samples in 150 bp paired-end mode, Eurofins Genomics typically achieves over 80% of base calls with a quality value higher than Q30 ( >99.9% accurate).
Do you guarantee a specific on-target output?
For samples that have successfully passed the initial quality check at Eurofins Genomics, we will guarantee the on-target output you have ordered. Average on-target coverage is calculated as the sum of the mapped bases at each target position, divided by the number of bases in the target (bases on-target / size of target region).
Which genes are enriched?
The target region corresponds to the bait coordinates of the Agilent SureSelectHuman All Exon V6 Kit: approx. 60 Mbp of exonic bases (>93% of the targets are covered with >20x, 90% of the genes are completely covered – all exons – 10% of the genes are partially covered)
Are all genes covered at 30x?
Due to varying efficiency of the enrichment baits (e.g., GC / AT content), targeted regions are covered differently and the range and uniformity of coverage varies over the target region. Eurofins Genomics applies the most recent Agilent Human All Exon kit design (V6) with improved design algorithms to better capture difficult regions and provide superior coverage uniformity.
What kind of quality controls do you perform?
The quality and quantity of each incoming sample will be determined by appropriate methods. Further quality controls are performed at various steps of the process.
Where should I send my samples?
Eurofins Genomics Europe Sequencing GmbH