InView™ Human Exon

The exome accounts for ~1% of the human genome and consists of functionally relevant coding regions (exons) which are most likely to cause differences in phenotypes. A versatile tool for discovering relevant variations is exome enrichment. In contrast to whole genome sequencing only 1-2% of the human genome needs to be sequenced.

Exome analyses allow a closer insight in these target regions providing comprehensive information for specific applications:
 

• Disease studies

• Pharmacogenomics

• Pharmacogenetics

• Clinical research

• Genetic predisposition analyses

• Evolutionary biology research (population-based studies within and between populations

GATC Biotech offers with InView^TM Human Exon a complete all-in-one service to support variant discovery for Mendelian diseases or population-based studies. The latest technologies for high throughput exome sequencing services are combined with streamlined workflows. A dedicated team of scientific project managers and bioinformaticians will ensure you receive reliable and reproducible results within fast turnaround times.

GATC Biotech uses the latest Agilent SureSelect^XT Human All Exon Enrichment V5 Kit allowing library preparation with subsequent exon enrichment. Sequencing is performed with Illumina’s HiSeq 2000, which is currently among Next Generation Sequencing platforms the most effective, providing the highest quality data. Regarding bioinformatics, GATC Biotech applies most comprehensive tools for data filtering to enable reliable detection of presumable variants.

Starting material                        

• Genomic DNA or various other sources like tissue, cells, blood and FFPE (Formalin-Fixed Paraffin Embedded) samples

• Optional Whole Genome Amplification (WGA) for low amount samples

Sequencing platform and enrichment

• Latest Illumina HiSeq 2000 system using 100 bp paired-end sequencing

• Agilent SureSelect^XT Human All Exon V5 Kit

Guaranteed sequencing coverage

• Average on target coverage of 30x, 60x or 120x

Bioinformatics

• Highly accurate discovery and annotation of SNPs and InDels, with information on e.g., gene ID, amino acid change, functional class and additional statistics relating to the variants

• Computational analyses through combination of different commercial and proprietary tools

• Results in common formats for visualisation and further analysis

• Results are delivered via your secured personal myGATC account

Optimised lab protocols combined with sophisticated parameters for data filtering and bioinformatics provide highly accurate data and reliable SNP and InDel analyses.

GATC Biotech has been a Next Generation Sequencing Service Provider since 2006

As Europe's leading sequencing service provider GATC Biotech has been developing sequencing and bioinformatics solutions for over twenty years. We have provided Next Generation Sequencing Services since 2006 and were the first to offer multiplatform sequencing approaches. GATC Biotech is heavily involved in many publicly funded projects and cooperates closely with well-known diagnostic institutes and the medical community in order to be up-to-date with current research and health topics.

ICGC Initiative: Sequencing for the International Cancer Genome Consortium

LifeCodexx: Clinical validation study for detection of fetal trisomy 21 using non-invasive test method

1. Where do I get my results and what do they look like?

All raw data as well as the analysed data can be downloaded via your secured myGATC account. Example analysed data of 30x coverage are available for download after login here:

Global Analysis_report.pdf

InDel Table

demo.AllExonV5.HAPMAP_30x.indel.bed

demo.AllExonV5.HAPMAP_30x.indel.tsv

demo.AllExonV5.HAPMAP_30x.indel.vcf

SNP table

demo.AllExonV5.HAPMAP_30x.snp.bed

demo.AllExonV5.HAPMAP_30x.snp.tsv

demo.AllExonV5.HAPMAP_30x.snp.vcf

TargetRegion 

SureSelect_AllExon_V5_Genes

SureSelect_AllExon_V5_hg19_target_coordinates

2. 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. To confidently discover variants in inhomogeneous samples like e.g., DNA extracted from normal and tumor cells, a higher overall coverage should be chosen. The amount of data needed to reach a certain coverage on the DNA of interest depends on the ratio normal/tumor DNA. Please note, that due to varying efficiency of the enrichment baits, the targeted regions are not covered evenly (see below).

3. 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 GATC Biotech. The 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 receive PCR duplicate rates of approx. 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.

4. Which and how much starting material should I send?

Raw data output guarantees currently only apply for freshly isolated, unamplified and pure human DNA samples (minimum 3 µg). Contamination by DNA of other species (> 3%; especially from closely related ones) 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 like e.g., Formalin-Fixed Paraffin Embedded (FFPE) samples is usually accepted. Please contact us if you need any details.

5. What do you mean by overlapping reads?

The fragmentation of genomic DNA according to the Agilent SureSelectXT protocol and following 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 200bp. Sequencing of those library fragments with 100bp paired end reads will generate partially overlapping reads which cover the same bases and hence create an artificial doubling of coverage at those positions. To assure accurate analyses those bases are excluded from the calculation of the average on target coverage and further downstream analyses. Furthermore GATC is continuously optimising its protocols to minimise the number of overlapping bases.

6. What is the quality of the data?

When sequencing human samples in 100bp paired-end mode GATC Biotech typically achieves over 80% of base calls with a quality value higher than Q30 ( >99.9% accurate).

7. Do you guarantee a certain on target output?

For samples which have successfully passed initial quality check at GATC Biotech, we will guarantee the on target output ordered by you. Average on target coverage is calculated as the sum of the mapped bases (without overlapping bases) at each target position, divided by the number of bases in the target (bases on-target / size of target region).

8. Which genes are enriched?

The target region corresponds to the bait coordinates of the Agilent SureSelect^XT Human All Exon V5 Kit (approx. 50 Mb, comprising 21.522 genes and 357.999 exons). Target region (bed file) of Exon V5 Kit are available here.

9. Are all genes covered with 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. GATC Biotech applies the most recent Agilent Human All Exon kit design (V5) with improved design algorithms to better capture difficult regions and increase coverage uniformity.

10. How much starting should I provide?

For optimal results we require at least 3 µg double-stranded, purified, high molecular and RNA-free DNA (concentration approx. 200 ng/µl; OD 260/280 ≥ 1.8; OD 260/230 ≥ 1.9).

11. What kind of quality controls do you perform?

The quality and quantity of each incoming sample will be determined by appropriate methods (e.g. agarose gel analysis / Qubit® Fluorometer / NanoDrop / Agilent 2100 Bioanalyzer). Further quality controls are performed at various steps of the process.

12. Where should I send my samples?

Please post your samples to:
GATC Biotech AG
European Genome and Diagnostics Centre
Jakob-Stadler-Platz 7
D-78467 Konstanz, Germany

Please don't use GATC Collection Points for shipment of NextGen samples because this will cause delay in sample arrival at the appropriate destination!